Biomatrix scaffolds for industrial scale dispersal

ABSTRACT

The present invention provides biomatrix scaffolds for industrial scale dispersal.

STATEMENT OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.14/813,993, filed Oct. 13, 2015, now U.S. Pat. No. 9,944,896, which is adivisional of U.S. patent application Ser. No. 13/807,206, filed Feb.12, 2013, now U.S. Pat. No. 9,102,913, which is the U.S. National Stageof PCT/US2011/042825, filed Jul. 1, 2011, which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.61/360,939, filed Jul. 2, 2010, the entire contents of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

Aspects of this invention were made with government support underNational Institutes of Health (NIH) Grant Nos. AA014243 andIP30-DK065933, National Institute of Diabetes and Digestive and KidneyDiseases (NIDDK) Grant No. DK34987, National Cancer Institute (NCI)Grant No. CA016086 and National Institute of Dental and CraniofacialResearch No. DE019569. The United States Government has certain rightsto this invention.

FIELD OF THE INVENTION

This invention concerns biomatrix scaffolds and methods of producingbiomatrix scaffolds and their use in diverse applications as intactscaffolds or as scaffolds that are sectioned or pulverized and dispersedin various ways for specific experimental and clinical uses.

BACKGROUND OF THE INVENTION

The ability to use differentiated cells ex vivo or in clinical programssuch as cell therapies depends on the ability to maintain the cells withan adult phenotype and fully functional or to be able to lineagerestrict stem cells or progenitors (“stem/progenitors”) to achieve thatadult phenotype. The ongoing revolution in stem cell research has madepossible the identification and isolation of stem/progenitor cellpopulations including those from embryonic, fetal and postnataltissues1. The ability to identify and isolate the stem/progenitors forall adult cell types and to expand and to differentiate them greatlyincreases the potential for utilizing them for pharmaceutical and otherindustrial research programs, for academic investigations and forclinical programs such as cell based therapies, and tissue engineering2.

Current methods for maintaining differentiated cells or of lineagerestricting stem cells to an adult fate ex vivo are partially successfuland involve plating the cells onto or embedded into a substratum of anextracellular matrix component(s) and into a medium comprised ofspecific hormones, growth factors and nutrients tailored for the adultphenotype desired. For very primitive stem cells such as embryonic stem(ES) cells or induced pluripotent stem (iPS) or postnatally-derived onesthat can go to multiple adult fates, such as mesenchymal stem cells(MSCs) or amniotic fluid-derived stem cells (AFSCs), the stem cells aresubjected to a mix of soluble signals and/extracellular matrixcomponents and must be treated with multiple sets of these signals overweeks of time. Typically the adult phenotype achieved is distinct withevery preparation and has over or under expression of certainadult-specific genes and/or aberrant regulation of one or more of theadult tissue-specific genes.

Extracellular matrix is secreted by cells, is adjacent to them on one ormore of their surfaces, and has long been understood to be thestructural support for cells7. It is an extraordinarily complex scaffoldcomposed of a variety of biologically active molecules that are highlyregulated and critical for determining the morphology, growth, anddifferentiation of the attached cells8. Tissue-specific gene expressionin cultured cells is improved by culturing the cells on matrix extractsor purified matrix components9. However, individual matrix components,alone or in combination, are unable to recapitulate a tissue's complexmatrix chemistry and architecture. This is related to the fact that thematrix components are in gradients associated with natural tissue zonesand with histological structures such as blood vessels. This complexityof the tissue matrix is more readily achieved by extractions thatdecellularize a tissue and leave behind the matrix as a residue10,11.However, current decellularization protocols result in major losses ofsome of the matrix components due to the use of matrix-degrading enzymesor buffers that solubilize matrix components.

The present invention provides biomatrix scaffolds and methods of makingand using such biomatrix scaffolds. The methods of this invention resultin the production of a tissue-specific extract enriched in a majority ofthe collagens of the tissue and with bound matrix components andmatrix-bound hormones, growth factors and cytokines that collectivelyyield more reproducible and potent differentiation effects on bothmature cells and in lineage restriction of stem/progenitor cellpopulations.

SUMMARY OF THE INVENTION

The present invention provides a method of producing a biomatrixscaffold from biological tissue for dispersal (e.g., industrial scaledispersal employing volumes as described, for example in the protocolsset forth in Example 2) onto culture apparatus, comprising: a) perfusingthe biological tissue or homogenizing the biological tissue with abuffer comprising a salt concentration from about 3.5M NaCl. to about4.5M NaCl.; b) perfusing the biological tissue or extracting thehomogenate of step (a) with a delipidating buffer comprising lipasesand/or detergents in a first medium, wherein the osmolality of saidfirst medium is from about 250 mOsm/kg to about 350 mOsm/kg and saidfirst medium is serum free and at neutral pH; then

c) perfusing the tissue or extracting the homogenate of step (b) with abuffer at a neutral pH and comprising a salt concentration from about2.0M NaCl. to about 5.0M NaCl., the concentration chosen to keepinsoluble collagens identified in the biological tissue; then

d) perfusing the tissue or extracting the homogenate of step (c) withRNase and DNase in a buffer; and then

e) rinsing the tissue or homogenate of step (d) with a second mediumthat is at neutral pH, is serum-free and has an osomolality from about250 mOsm/kg to about 350 mOsm/kg, thereby producing an intact orhomogenized biomatrix scaffold from the biological tissue, saidbiomatrix scaffold comprising at least 95% of the collagens and mostcollagen-associated matrix components and matrix bound growth factors,hormones and cytokines of the biological tissue;

f) diluting the biomatrix scaffold in basal medium;

g) freezing the biomatrix scaffold of (f) at about −80° C.;

h) pulverizing the biomatrix scaffold of (g) by cryogenic grinding intobiomatrix particles ranging in size from about 1 μm to about 100 μm;

i) thawing the biomatrix particles of (h) in suspension in basal medium;and

j) dispersing the biomatrix particles of step (i) onto a cultureapparatus, thereby producing a biomatrix scaffold from biological tissuefor dispersal (e.g., industrial scale dispersal as described herein)onto culture apparatus.

The present invention further provides biomatrix scaffold produced bythe methods of this invention.

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to other embodiments describedherein. It should be appreciated that the invention can be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panels A-E1. Rat liver biomatrix scaffold preparation. (A)Four-step decellularization process comprised of perfusion wash,delipidation with PLA2 and SDC, high salt washes, and nuclease treatmentfor nucleic acid removal. (B-D) Four stages in the preparation of ratbiomatrix scaffold. (B) After perfusion wash with basal medium for 10minutes the liver becomes pale; (C) during delipidation, the liverbecomes partially transparent under GC (D) final intact scaffold lookstransparent at 40 minutes of perfusion; (E) biomatrix scaffold shown atlow magnification. (E1) Visualization of scaffold perfused withrhodamine-labeled dextran particles demonstrates progressive flow fromlarge vessels to the fine blood vessel branches along the channelswithout leakage, indicating patent vasculature in scaffolds.Corresponding hematoxylin and eosin (H&E) staining of biomatrix scaffoldin different stages demonstrated that the histological structures suchas blood vessels and lace-like matrix enveloping the parenchyma arepreserved, whereas cells are removed. The normal rat hepatic portaltriad structure consisting of the portal vein (PV), hepatic artery (HA),and bile duct (BD) (B1); the matrix fibers becoming apparent as thecells are gradually removed during the decellularization process (C1);decellularized portal triad region, compare (B1) with that in (D1), D2and D3 show that all of the cells are removed from the matrix scaffoldbut mesh structures are preserved such as the blood vessels, GC, and thelace-like matrix that surrounds muralia of parenchymal cells.

FIG. 2, Panels A-H. TEM (A-C) and SEM (D-H) images of rat liverbiomatrix scaffolds. (A) Low magnification of blood vessel (BV) with athick wall (W). Collagen Type I (large arrowhead) is numerous andcontains cross-sections of individual fibers that do not take up heavymetal stains (white dots, small arrowheads). (B) Higher magnification ofa vessel wall shows basement membrane (large arrow), amorphous elastin(*) and associated elastic fibers, a rare membrane vesicle remnant(small arrowhead), a collagen Type I banded fiber (arrowhead) and smallfibrils (small arrows). The small fibrils are probably fibrillin (TypeVI collagen) that associates closely with and helps organize Type Icollagen. (C) High magnification of Type I collagen with 64 nm bandingpattern (arrows). (D) Low magnification of a vessel with thin wall (BV)and the wall of a larger vessel (W). (E) At higher magnification, thelarge vessel wall (W) is scalloped, consistent with hepatic artery of aportal triad, see (A). Beneath the wall are numerous Type I collagenbundles (large arrow) linked by long branching thin, reticular (TypeIII) collagen fibrils (small arrows). (F) A large bundle of Type Icollagen has characteristic parallel fibers (large arrow) associatedwith a variety of smaller fibers (arrow) and nodular or beaded fibers(arrowhead). (G) 3D-meshwork of large/small fibers interlinked in aplane that firms a boundary such as to a liver sinusoid. (H) Highermagnification of the meshwork showing a variety of fibers (arrows): TypeIII collagen (larger diameter straight), elastic fibers or Type VIcollagen.

FIG. 3, Panels A-B. Chemical analysis of collagens and expression ofextracellular matrix (ECM) components in biomatrix scaffolds. (A) Thecontent of three amino acids, all found in collagens: hydroxyproline(Hyp), hydroxylysine (Hyl), and glycine (Gly). The numbers represent theresidues of each amino acid/1,000 amino acids. The data indicate thedramatic increase in the collagen content in the decellularizationprocess going from <0.2% in liver to more than 15% in the biomatrixscaffolds. (B) Immunohistochemical staining of matrix molecules inbiomatrix scaffolds, shows distribution in liver biomatrix scaffolds oflaminin (LAM), heparan sulfate (HS), collagen type III (COL3) andfibronectin (FN) and typical basement membrane proteins in associationwith remnants of blood vessels. At higher magnification, one can observemain members of basement membrane, including type IV collagen (COL4),entactin (Ent; also called nidogen), laminin (LAM) and perlecan (Per), aform of HS-PG in the portion of the scaffolds near the portal triads.

FIGS. 4A-D. Pattern of ECM components from portal triad to central veinin biomatrix scaffolds. Histological comparison from portal triad(zone 1) to central vein (zone 3) of normal liver (A) and liverbiomatrix scaffold (B); both are hematoxylin/eosin stained sections. (C)The model illustrating a stem cell and maturational lineage system inthe liver with representative matrix components shown that form patternsassociated with the liver zonation. The components are listed in orderof abundance from the findings of immunohistochemistry. The knownlineage stages within human livers begin periportally in zone 1 (aroundportal triads) and progress in maturation ending with apoptotic cells inzone 3. The known matrix chemistry identified in the liver's stem cellniche is comprised of hyaluronans, type III collagen, a form of lamininthat binds to cx6134 integrin, and a weakly sulfated form of CS-PG43,44.Just outside the stem cell niche are found Type IV collagen, normallysulfated CS-PGs and HS-PGs and forms of laminin binding to cx6134integrin. HP-PGs have been documented to be located uniquelypericentrally45,46. (D) The survey of matrix components and theirlocation in liver versus those in biomatrix scaffolds, data summarizedfrom immunohistochemistry findings (N/D=not tested. *Found by others tobe exclusively near central veins). Most components of the cytoskeletonare lost during the washes, residues of some, but not all, cytoskeletalproteins are present. The scaffolds are devoid of detectable amounts oftubulin, desmin, and actin (phalloidin assays). However, there are traceamounts of cytokeratins scattered randomly in the scaffolds; traceamounts of a-smooth muscle actin around remnants of blood vessels at theportal triads; and low levels of vimentin throughout.

FIG. 5, Panels A-I. Characterization of hHpSCs on liver biomatrixscaffolds versus on type I collagen. Phase-contrast images (A-D) showthe morphologic changes of hHpSC colonies derived from the same liverand cultured in serum-free Kubota's medium and on tissue culture plastic(A), one of the conditions for self-replication, versus in thedifferentiation conditions of the serum-free differentiation medium forliver, and on type I collagen (B) versus on bovine liver biomatrixscaffolds (C-E). Functional and fully viable cultures did not last morethan 2 weeks on type I collagens. By contrast, those on the liverbiomatrix scaffolds were viable and healthy and with a full repertoireof functions lasting at least a month. The cultures transitioned tocells by days 7-12 with increased cytoplasmic/nuclear ratio and markedglycogen expression (C) and then to ones with classic polygonalhepatocyte morphology interspersed by clear bile canaliculi (D), aculture morphology that persisted thereafter, as indicated in therepresentative culture at day 24 (E). RT-PCR assays show gene expressionchanges of hHpSCs under self-replication conditions on culture plastic(F) versus on rat liver biomatrix scaffolds on day 7 (G). We comparedexpression of hHpSC markers, including CXCR4 and EpCAM; earlyhepatocytic genes including CK19 (KRT19), HNF6, FOXA2, APP and lowlevels of albumin; mature hepatocytic markers including high levels ofalbumin (ALB), transferrin (TF), CYP450-3A4, tyrosine aminotransferase(TAT), and glucose-6-phoshatase (G6PC) and cholangiocytic genes,including CFTR, gamma glutamyl transpeptidase (GGT1), anion exchange 2(AE2) and apical sodium-dependent bile acid transporter (ASBT).Biochemical assays measuring urea (H) synthesized in cultures on type Icollagen versus on rat liver biomatrix scaffolds and CYP450-3A4 activity(I) in cultures on type I collagen versus on biomatrix scaffoldsprepared from either rat or bovine livers. Table 7 provides a summary ofquantitative measures comparing attachment, viability, growth, culturelife span, and tissue-specific gene expression of hHpSCs freshlyisolated, under culture conditions for self-replication (type IIIcollagen), or under conditions for differentiation on collagen I, versusliver biomatrix scaffolds.

FIG. 6, Panels A-D. Immunofluorescence staining of cells lineagerestricted from hHpSCs on biomatrix scaffolds. (A) Stained with hepaticspecific marker:albumin (Alb, light grey) and hepatic stem cell surfacemarker: EpCAM (white). Note that cells plated on biomatrix scaffold donot express EpCAM. Scale bar=200 μm. (B) Stained with early hepaticmarker a-fetoprotein (APP, light grey) and with an antibody to humancholangiocyte marker, cytokeratin 19 (CK19, white) that at this level ofexpression is indicative of mature cholangiocytes. The antibody to CK19assay is human-specific and did not stain the residue at rat CK19 in thescaffolds not seeded with cells. The APP expression is low but stillevident at day 7. Scale bar=200 μm. (C) Stained with Alb (light grey)and hepatic stellate cell marker, a-smooth muscle actin (ASMA, white).The expression of albumin and ASMA is a strong indication that bothmaturing hepatocytes and stellate cells are present. Scale bar=100 μm.(D) Stained with functional hepatic protein CYP450-3A4 (light grey) andcholangiocyte-specific marker, secretin receptor (SR, white) showingthat the maturing hepatocytes and cholangiocytes are functional andexpress classic markers for these two cell types. Scale bar=200 μm.

FIG. 7, Panels A-D. Stability of fully functional, mature humanhepatocytes on biomatrix scaffolds. Adult human hepatocytes plated inthe differentiation medium and onto type I collagen (A,B) versus onbovine liver biomatrix scaffolds (C) that were cryogenically pulverized,dispersed in medium and allowed to sediment onto the plates. Cells ontype I collagen are fully viable and at their peak of differentiationfrom 7-12 days (A-shown at 7 days); they begin to deteriorate after 2weeks, and by 20 days (B) they are dead, dying and non-functional. Bycontrast, those plated onto liver biomatrix scaffolds (C) are functionalfor at least 8 weeks (longer times have not been assessed yet); here isshown after 21 days in culture on pulverized liver biomatrix scaffolds.CYP450-3A4 assays on cultures of two separate preparations ofcryopreserved adult human liver cells plated onto biomatrix scaffoldsversus on type I collagen and assayed on day 12 (D). The sample ZHep-007is representative of cryopreserved samples with good attachment afterthawing; the sample ZL-013 is representative of those lots that havepoor or no attachment after thawing. Thus, even these poorer qualitysamples are able to attach to biomatrix scaffolds and remain viable longterm. In both samples assayed, the levels of P450s are higher whencultured on liver biomatrix scaffolds. With time on the biomatrixscaffolds, the lots of poorer quality cryopreserved cells will improve.

FIG. 8. Activation of phospholipase A2 by sodium deoxycholate to producelysolecithin. Principle of the protocol: Phospholipase A2 (PLA2)activated by sodium deoxycholate will degrade the phosphoglyceridelocated on the cytoplasmic membrane and mitochondrial membrane intolysolecithin, a powerful surfactant, which induces cell necrosis.

FIG. 9. Analysis of the collagen composition of rat livers versus ratliver biomatrix scaffolds. The amino acid composition of biomatrix(black) and whole liver (light grey) presented in the form of a RoseDiagram. A three-letter abbreviation is used for each amino acidanalyzed. Tryptophan and cysteine were not analyzed. The numbersindicate the amino acid residues/1,000.

FIG. 10, Panels A-C. Nucleic acid analysis of rat liver biomatrixscaffold. Phase contrast photo (A) and fluorescent DAPI staining (B) ofthe liver biomatrix slide, and quantitative assays on total DNA and RNAfrom rat fresh liver tissue versus biomatrix scaffold (C).

FIG. 11, Panels A-C. Staining of the biomatrix scaffold after platinghHpSCs onto the biomatrix scaffold. Live (calcein-AM, white)/Dead(ethidium bromide or EtD-Br1, light grey) assay indicates hHpSCscolonies were viable on biomatrix scaffold sections but did not take updye in the middle of the colonies (A,B) for the first few days due tothe known pumps in the stem cells (e.g., MDR1) that eliminate the vitaldyes. In (B) the fluorescence image is merged with the phase one toindicate that the center of the colony contains cells. By day 7, thecells throughout the colony had differentiated and took up the vital dyein almost all the cells throughout the colony (C).

FIG. 12, Panels A-F. Comparison of rat hepatocytes cultured on type Icollagen to rat hepatocytes cultured on rat liver biomatrix scaffolds.Adult rat hepatocytes cultured on type I collagen and biomatrix scaffoldat day 3 (A,C) and day 10 (B,D). They attached within several minutes onliver biomatrix scaffolds and survived for as long as tested, more than8 weeks (C,D); longer time periods were not tested. The cultures arevery three-dimensional on the biomatrix scaffolds. Urea synthesis (E)and cell viability assay (F) at day 1, 3, 5, 7, 10, 14, 21 and 28, n=3.

FIG. 13, Panels A-D. Comparison of a human pancreas to a humanpancreatic biomatrix scaffold. Human pancreas (A) vs human pancreaticbiomatrix scaffold (B-D) embedded in paraffin, sectioned and stainedwith Hematoxylin and Eosin (H&E). Islet structures have been outlined inB. The acinar regions of pancreatic biomatrix scaffolds are shown in Cand D.

FIG. 14. Comparison of representative matrix components and onecytoskeletal component, vimentin, found in human pancreatic tissueversus rat pancreatic biomatrix scaffolds. Other cytoskeletal components(desmin, tubulin, actin) were not found in detectable amounts or werefound in trace amounts (cytokeratins). The dashed lines encircle islets,note that syndecan 1 and collagen type VI are strongly positive in theislets both in pancreas tissue and in biomatrix scaffolds. Syndecan 1 isfound only in the islets (dashed line) but not in the acinar cells(arrows); collagen type III is more enriched in acinar cells and aroundblood vessels (arrows), but not in the islets.

FIG. 15, Panels A-C and COL1-HS. Histological and immunohistochemistrystaining of human duodenum biomatrix scaffold. (A) Outside and lumenside of human duodenum biomatrix scaffold. The multilayer structuresbetween the normal tissue (B) and biomatrix scaffold (C) were comparedin H&E stained sections and results show scaffolds retained the villusand blood vessels in the mucosa and submucosa layers. Lower panelsCOL1-HS show immuohistochemistry staining of human duodenum biomatrixscaffold indicated variable amounts of extracellular matrix proteinsretained in the scaffold.

FIG. 16, Panels A-D. Comparison of a human gallbladder tissue to a humangallbladder biomatrix scaffold. Human gallbladder tissue (A,B) versusbiomatrix scaffolds (C,D) prepared from it. The tissue and the biomatrixscaffolds were embedded in paraffin, sectioned and stained withhematoxylin and eosin.

FIG. 17. Example of preparation of biomatrix solution for 1:24 dilution.30 ml×3=90 ml. 30 ml biomatrix+90 ml Solution 5=120 ml biomatrixsolution for 1:24 dilution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP § 2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration (e.g., the percentage of collagen inthe total proteins in the biomatrix scaffold) and the like, is meant toencompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

The present invention is directed to the discovery and development of abiomatrix scaffold that has unexpected improvements and advantages overdecellularized tissue scaffolds now known, some examples of theimprovement and advantage being the use of the biomatrix scaffold ofthis invention to efficiently maintain mature cells and/or to lineagerestrict and/or differentiate stem cells to mature fates and/or tomaintain such matured cells as functional for an extended period oftime. As a further example, use of the biomatrix scaffolds of thisinvention reduces the time to produce cells of mature fates from aboutthree to six weeks or more to about one to two weeks. The biomatrixscaffolds of this invention are produced using specific protocols thatemploy the appropriate balance of salt concentration and ionic strength(different collagens have different solubility constants (23)) for agiven tissue, to allow for the retention of native collagens present inthat tissue in insoluble form, resulting in a biomatrix scaffold thatretains a high percent of native collagens that provide signals to drivelineage restriction and differentiation. In contrast, decellularizedscaffolds produced according to known protocols do not employ such abalance of salt concentration and ionic strength to allow for retentionof a high percent of these native collagens and most of these nativecollagens are lost when these known protocols are used. Furthermore, thebiomatrix scaffolds of this invention allow for production of lineagedependent (e.g., differentiation dependent) viruses and/or pathogens inamounts sufficient for experimental and/or therapeutic use (e.g., forvaccine production).

Thus, in one embodiment, the present invention provides a method ofproducing a biomatrix scaffold from biological tissue, comprising thesteps of: a) perfusing the biological tissue or homogenizing thebiological tissue with a first medium, wherein the osmolality of saidfirst medium is from about 250 mOsm/kg to about 350 mOsm/kg and saidfirst medium is serum free and at neutral pH; then b) perfusing thebiological tissue or extracting the homogenate of step (a) with adelipidating buffer comprising lipases and/or detergents in said firstmedium; then c) perfusing the tissue or extracting the homogenate ofstep (b) with a buffer at a neutral pH and comprising a saltconcentration from about 2.0M NaCl. to about 5.0M, the concentrationchosen to keep insoluble collagens identified in the biological tissue;then d) perfusing the tissue or extracting the homogenate of step (c)with RNase and DNase in a buffer; and then e) rinsing the tissue orhomogenate of step (d) with a second medium that is at neutral pH, isserum-free and has an osomolality from about 250 mOsm/kg to about 350mOsm/kg, thereby producing an intact or homogenized biomatrix scaffoldfrom the biological tissue, said biomatrix scaffold comprising at least95% (e.g., 80%, 85%, 90%, 95%, 98%, 99%, 100%) of the collagens and mostcollagen-associated matrix components and matrix bound growth factors,hormones and cytokines of the unprocessed biological tissue. Alsoprovided herein is a biomatrix scaffold produced by any of the methodsof this invention.

“Biomatrix scaffold” as used herein refers to an isolated tissue extractenriched in extracellular matrix, as described herein, which retainsmany or most of the collagens and collagen-bound factors found naturallyin the biological tissue. In some embodiments essentially all of thecollagens and collagen-bound factors are retained and in otherembodiments the biomatrix scaffold comprises all of the collagens knownto be in the tissue. The biomatrix scaffold may comprise at least about50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% ofthe collagens, collagen-associated matrix components, and/or matrixbound growth factors, hormones and/or cytokines, in any combination,found in the natural biological tissue. In some embodiments thebiomatrix scaffold comprises at least 95% of the collagens and most ofthe collagen-associated matrix components and matrix bound growthfactors, hormones and/or cytokines of the biological tissue. Asdescribed herein, “most of the collagen-associated matrix components andmatrix bound growth factors, hormones and/or cytokines of the biologicaltissue” refers to the biomatrix scaffold retaining about 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% of thecollagen-associated matrix components and matrix bound growth factors,hormones and/or cytokines found in the natural (e.g., unprocessed)biological tissue.

Exemplary collagens include all types of collagen, such as but notlimited to Type I through Type XXIX collagens. The biomatrix scaffoldmay comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, 99.5% or more of one or more of the collagens found in thenatural biological tissue and/or may have one or more of the collagenspresent at a concentration that is at least about 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more of that found in thenatural biological tissue. The amount of collagen in the biomatrixscaffold can be determined by various methods known in the art and asdescribed herein, such as but not limited to determining thehydroxyproline content.

Exemplary collagen-associated matrix components include, but are notlimited to, adhesion molecules; adhesion proteins; L- and P-selectin;heparin-binding growth-associated molecule (HB-GAM); thrombospondin typeI repeat (TSR); amyloid P (AP); laminins; nidogens/entactins;fibronectins; elastins; vimentins; proteoglycans (PGs); chondroitinsulfate-PGs (CS-PGs); dermatan sulfate-PGs (DS-PGs); members of thesmall leucine-rich proteoglycans (SLRP) family such as biglycan anddecorins; heparin-PGs (HP-PGs); heparan sulfate-PGs (HS-PGs) such asglypicans, syndecans, and perlecans; and glycosaminoglycans (GAGs) suchas hyaluronans, heparan sulfates, chondroitin sulfates, keratinsulfates, and heparins. In some embodiments the biomatrix scaffoldcomprises, consists of, or consists essentially of collagens,fibronectins, laminins, nidogens/entactins, elastins, proteoglycans,glycosaminoglycans, growth factors, hormones, and cytokines (in anycombination) bound to various matrix components. The biomatrix scaffoldmay comprise at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 99.5% or more of one or more of the collagen-associated matrixcomponents, hormones and/or cytokines found in the natural biologicaltissue and/or may have one or more of these components present at aconcentration that is at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, 99.5% or more of that found in the natural biologicaltissue. In some embodiments the biomatrix scaffold comprises all or mostof the collagen-associated matrix components, hormones and/or cytokinesknown to be in the tissue. In other embodiments the biomatrix scaffoldcomprises, consists essentially of or consists of one or more of thecollagen-associated matrix components, hormones and/or cytokines atconcentrations that are close to those found in the natural biologicaltissue (e.g., about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%of the concentration found in the natural tissue).

Exemplary growth factors include, but are not limited to, fibroblastgrowth factors (FGFs), nerve growth factors (NGFs), epidermal growthfactors (EGFs), transforming growth factors, hepatocyte growth factors(HGFs), platelet-derived growth factors (PDGFs), insulin-like growthfactors (IGFs), IGF binding proteins, basic fibroblast growth factors,and vascular endothelial growth factors (VEGF). Exemplary cytokinesinclude, but are not limited to interleukins, lymphokines, monokines,colony stimulating factors, chemokines, interferons and tumor necrosisfactor (TNF). The biomatrix scaffold may comprise at least about 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more(in any combination) of one or more of the matrix bound growth factorsand/or cytokines found in the natural biological tissue and/or may haveone or more of these growth factors and/or cytokines (in anycombination) present at a concentration that is at least about 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more of thatfound in the natural biological tissue. In some embodiments thebiomatrix scaffold comprises physiological levels or near-physiologicallevels of many or most of the matrix bound growth factors, hormonesand/or cytokines known to be in the natural tissue and/or detected inthe tissue and in other embodiments the biomatrix scaffold comprises oneor more of the matrix bound growth factors, hormones and/or cytokines atconcentrations that are close to those physiological concentrationsfound in the natural biological tissue (e.g., differing by no more thanabout 30%, 25%, 20%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% incomparison). The amount or concentration of growth factors or cytokinespresent in the biomatrix scaffold can be determined by various methodsknown in the art and as described herein, such as but not limited tovarious antibody assays and growth factor assays.

“Biological tissue” as used herein refers to any tissue of a living ordeceased organism or any tissue derived from a living or deceasedorganism. The term “natural biological tissue” and variations thereof asused herein refer to the biological tissue as it exists in its naturalor unmodified state in the organism. The biomatrix scaffolds of thepresent invention can be prepared from any biological tissue. Thebiological tissue may include any single tissue (e.g., a collection ofcells that may be interconnected) or a group of tissues making up anorgan or part or region of the body of an organism. The tissue maycomprise a homogeneous cellular material or it may be a compositestructure such as that found in regions of the body including the thoraxwhich for instance can include lung tissue, skeletal tissue, and/ormuscle tissue.

Exemplary biological tissues of this invention include, but are notlimited to liver, lung, thyroid, skin, pancreas, blood vessels, bladder,kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein,heart and intestines, including any combination thereof. The organism(i.e., subject) from which the biological tissue is associated with orderived from may be any animal, including mammals and non-mammals suchas invertebrates.

Exemplary subjects include, but are not limited to mammals, such as butnot limited to, humans, mice, rats, ferrets, hamsters, rabbits, guineapigs, pigs, porcine, dogs, cats, horses, cows, sheep, monkeys, andchimpanzees, and non-mammals, such as but not limited to birds,reptiles, and invertebrate animals. The subject may be any age and/orsize. The biological tissue may be healthy, diseased, and/or havegenetic mutations. In some embodiments the biomatrix scaffolds of thepresent invention are tissue specific in their chemistry andfunctionality, i.e., the biomatrix scaffolds are representative orcomparable to the biological tissue from which they were created interms of their chemistry and functionality.

In some embodiments the native histology and patent vasculatures aremaintained in the biomatrix scaffolds. This may include the recognizableremnants of major histological entities of the biological tissue, suchas but not limited to blood vessels and other vasculature for anytissue; bile ducts and Glisson's capsule (GC) of the liver; pancreaticducts, islets and acini of the pancreas; bronchi, trachea, and alveoliof the lungs, etc. In other embodiments the biomatrix scaffold'schemistry is matched to the histology (e.g., matrix around blood vesselsis distinct from that around hepatocytes). In some embodiments thechemistry of the biomatrix scaffold is in a gradient that is correlatedwith the histology. For example, when the biological tissue is theliver, the biomatrix scaffold may retain the gradient in the matrixchemistry correlating with the hepatic acinar zones 1-3 from portaltriad to central vein and with histological entities such as vascularchannels and Glisson's capsule (GC). Further examples include, but arenot limited to, blood vessels where the chemistry of the matrix aroundthe blood vessels is replete with high levels of network collagens(e.g., type IV and type VI), elastins, and forms of HS-PGs; around thehepatocytes in the periportal zone (zone 1), where laminins are high inconcentration along with a mix of CS-PGs and HS-PGs, whereas around thepericentral zone (zone 3), are hepatocytes surrounded by a mix of HS-PGsand HP-PGs; associated with the bile ducts where there are high levelsof type I collagen, fibronectins and forms of CS-PGs and DS-PGs. Thereare parallel gradients in matrix chemistry in every tissue.

There are a number of rinse media, such as the first and second medium,and buffers that may be utilized in the present invention. Inparticular, any rinse medium or buffer may be used that maintains thecollagens and bound factors (e.g., matrix components, growth factors,and cytokines) in an insoluble state. When choosing a medium or buffer,the salt concentration, pH, and ionic strength should be suitable tomaintain the collagens and/or most or many of the collagen-bound matrixcomponents and other factors (e.g., by virtue of their chemicalconnections directly or indirectly with the collagens) in an insolublestate. Table 1 provides molar concentration ranges of sodium chloridefor various types of collagen to aid one of ordinary skill in the art inproviding media and buffers that ensure the collagens,collagen-associated matrix components, and matrix bound growth factorsand cytokines remain insoluble. Deyl et al. (“Preparative procedures andpurity assessment of collagen proteins” Journal of Chromatography B 790(2003) 245-275) additionally provides information on collagen chemistrythat can facilitate identification of the optimal conditions formaintaining collagens and bound factors in an insoluble state and isincorporated herein by reference in its entirety.

Table 1 demonstrates that pH is a variable working in conjunction withsalt concentration to define solubility. By having high saltconcentrations, the pH can be neutral. In some embodiments of thepresent invention, the salt concentration chosen is one that maintainsall the collagens of the tissue in an insoluble state, not just one ofthe collagens of the tissue in an insoluble state. For example, theknown collagens in fetal liver are ones that are insoluble in saltconcentrations of about 4.5M NaCl. and those in adult liver tissue thatare insoluble in salt concentrations of about 3.4M-3.5M NaCl.

The osmolality of any of the rinse media and/or buffers may be, forexample, from about 200 mOsm/kg to about 400 mOsm/kg, from about 250mOsm/kg to about 350 mOsm/kg, from about 275 mOsm/kg to about 325mOsm/kg, or from about 300 mOsm/kg to about 325 mOsm/kg, includingwithout limitation any values within these ranges not explicitly recitedherein. Distilled water and dilute buffers (e.g., with osmolality <100mOsm/kg) will result in the loss of significant amounts of collagen,collagen-associated matrix components and matrix bound growth factorsand cytoldnes. Thus, in some embodiments of the methods of thisinvention, distilled water and dilute buffers are not included.

As one of ordinary skill in the art would recognize, osmolality is anexpression of solute osmotic concentration per mass, whereas osmolarityis per volume of solvent. Thus, conversion from osmolarity to osmolalitycan be made by multiplying by the mass density. Osmolality can bemeasured using an osmometer which measures colligative properties, suchas freezing-point depression, vapor pressure, and boiling-pointelevation.

Osmolarity is the measure of solute concentration, defined as the numberof osmoles (Osm) of solute per liter (L) of solution (osmol/L or Osm/L).The osmolarity of a solution is usually expressed as Osm/L. Whereasmolarity measures the number of moles of solute per unit volume ofsolution, osmolarity measures the number of osmoles of solute particlesper unit volume of solution. Osmolality is a measure of the osmoles ofsolute per kilogram of solvent (osmol/kg or Osm/kg).

Molarity and osmolarity are not commonly used in osmometry because theyare temperature dependent. This is because water changes its volume withtemperature. However, if the concentration of solutes is very low,osmolarity and osmolality are considered equivalent.

The osmolarity of a solution can be calculated from the followingexpression:

osmol/L=I:i.fJi n.ioi

where rp is the osmotic coefficient, which accounts for the degree ofnon-ideality of the solution; n is the number of particles (e.g. ions)into which a molecule dissociates; C is the molar concentration of thesolute; and the index i represents the identity of a particular solute.In the simplest case (jJ is the degree of dissociation of the solute.Then, rp is between 0 and 1 where 1 indicates 100% dissociation.However, rp can also be larger than 1 (e.g., for sucrose). For salts,electrostatic effects cause rp to be smaller than 1 even if 100%dissociation occurs.

Perfusion of the biological tissue with any medium or buffer may beaccomplished by forcing the medium or buffer through the relevantvasculature of the biological tissue. For example, if the biologicaltissue is the liver, then the medium or buffer may be perfused throughthe portal vein of the liver. Alternatively, the medium or buffer may bepoured over the biological tissue and/or allowed to diffuse through thebiological tissue. For example, the biological tissue may be submergedand/or dialyzed in the medium or buffer allowing the medium or buffer todiffuse through the biological tissue. While submerged and/or dialyzedin the medium or buffer the solution and biological tissue may beshaken, such as on a rocker, and/or stirred. In some embodiments themedia and buffers are perfused through the relevant vasculature of thebiological tissue.

Alternatively, the tissue may be homogenized in the initial medium andthe buffers and media used thereafter being for extraction of thehomogenate. The homogenized versions of the biomatrix scaffolds areprepared from large organs (e.g., from cow or pig tissues), are thenpulverized into powder at liquid nitrogen temperatures, and the powderused on dishes for culture studies.

In some embodiments the first medium and/or the second medium is a basalmedium, such as but not limited to RPMI 1640, DME/F12, DME, F12, BME,DMEM, Waymouth's, or William's medium. Other exemplary basal media areknown in the art and are commercially available. The first medium and/orsecond medium can comprise, consist essentially of or consist ofcomponents that are combined to keep most collagens insoluble and asnative molecules, as described herein (e.g., by the particularcombination of osmolality and ionic strength as well as the absence ofserum). The first medium and/or second medium may comprise, consist of,or consist essentially of constituents present or similar to ormimicking those present in the interstitial fluid such as but notlimited to water; salts such as but not limited to inorganic salts;vitamins; minerals; amino acids such as but not limited to glycine,serine, threonine, cysteine, asparagine, and/or glutamine; sugars; fattyacids; coenzymes; hormones; and neurotransmitters. In certainembodiments where the first medium and/or second medium comprisesconstituents present or similar to or mimicking those present in theinterstitial fluid, the constituents can yield an osmolalityapproximately equivalent to the osmolality of commercially availablebasal medium or yield an osmolality from about 250 mOsm/kg to about 350mOsm/kg. In some embodiments the first medium and/or second mediumincludes media that are serum free, comprise constituents present ininterstitial fluid, and/or have an osmolality from about 250 mOsm/kg toabout 350 mOsm/kg. Such media can also be at neutral pH. The specificcomposition of the first medium and/or second medium is determined, inparticular embodiments, by the insolubility constants of the collagensof the biological tissue used to make the biomatrix scaffold, as wouldbe known to one of ordinary skill in the art.

The delipidating buffer of the present invention should be effective andyet gentle. The delipidating buffer may comprise, consist of, or consistessentially of detergents or surfactants, basal medium, salts, and/orlipases. When choosing components for the delipidating buffer, harshdetergents (e.g., sodium dodecyl sulfate; TritonX-100) should be avoidedto minimize loss of matrix components. Exemplary detergents of thisinvention include but are not limited to anionic detergents, such assalts of deoxycholic acid, 1-heptanesulfonic acid, N-laurylsarcosine,lauryl sulfate, 1-octane sulfonic acid and taurocholic acid; cationicdetergents such as benzalkonium chloride, cetylpyridinium,methylbenzethonium chloride, and decamethonium bromide; zwitterionicdetergents such as alkyl betaines, alkyl amidoalkyl betaines,N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, andphosphatidylcholine; and non-ionic detergents such as n-decyla-D-glucopyranoside, n-decyl 13-D-maltopyranoside, n-dodecyl13-D-maltoside, n-octyl 13-D-glucopyranoside, sorbitan esters,n-tetradecyl 13-D-maltoside, tritons, Nonidet-P-40, Poloxamer 188, andany of the Tween group of detergents; sodium lauryl sulfate; and sodiumdeoxycholate. In some embodiments the delipidating buffer comprisessodium deoxycholate.

Exemplary lipases include, but are not limited to, phospholipases suchas phospholipase A2, human pancreatic lipase, sphingomyelinases,lysosomal lipase, endothelial lipase, and hepatic lipase. In someembodiments the delipidating buffer comprises phospholipase A2. In otherembodiments the delipidating buffer comprises sodium deoxycholate andphospholipase A2. This combination, in some embodiments, can comprisefrom about 20 to about 50 units/L phospholipase A2 and about 1% sodiumdeoxycholate prepared in a basal medium of neutral pH and serum-free,which can be for example, the first medium. The combination of sodiumdeoxycholate and phospholipase A2 rapidly degrades the phosphoglyceridelocated on the cytoplasm membrane and mitochondrial membrane intolysolecithin, a powerful surfactant, which can induce necrosis andcytolysis. As one of ordinary skill in the art would recognize, theamount and type of lipase and/or detergent may depend on the biologicaltissue.

The step of perfusing the biological tissue with the delipidating bufferis carried out, in some embodiments, until the tissue becomestransparent. In other embodiments the step of perfusing the biologicaltissue with the delipidating buffer is carried out until the effusionbecomes clear. In some embodiments the delipidating step is carried outuntil the tissue becomes transparent and the effusion becomes clear.

In some embodiments prolonged exposure of the biomatrix scaffolds toenzymes from the disrupted cells is avoided since it can greatlydecrease the content of elastin and the content of glycosaminoglycanssuch as heparan sulfates, chondroitin sulfates, dermatan sulfates andheparins, which are sites at which cytokines and growth factors bind.Exposure to the enzymes from the disrupted cells may be avoided, forinstance, during delipidation and/or the subsequent washes afterdelipidation. In some embodiments, use of a protease inhibitor and/orcareful control of the pH, temperature, and/or time can be employed tolimit the activity of the proteases and/or other enzymes from disruptedcells.

Exemplary protease inhibitors include but are not limited to serineprotease inhibitors such as but not limited to antipain, aprotinin,chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF,TLCK, TPCK, leupeptin and soybean trypsin inhibitor; cysteine proteasessuch as but not limited to IAA (indoleacetic acid) and E-64; asparticprotease inhibitors such as but not limited to pepstatin and VdLPFFVdL;metalloproteases such as but not limited to EDTA, 1,10-phenanthrolineand phosphoramodon; exopeptidases such as but not limited to amastatin,bestatin, diprotin A and diprotin B; thiol proteases; a-2-macroglobulin,soybean or lima bean trypsin inhibitor; pancreatic protease inhibitor;egg white ovostatin; egg white cystatin; and combinations of proteaseinhibitors, commonly referred to as a “protease inhibition cocktail” bycommercial suppliers of such inhibitors.

The pH of the biomatrix scaffold, buffers, and/or media can bemaintained at from about 6.0 to about 9.0, from about 6.5 to about 8.5,from about 7.0 to about 8.0, or from about 7.5 to about 8.0. In someembodiments, the biomatrix scaffold, buffers, and/or media aremaintained at a pH of from about 7.5 to about 8.0 or are maintained at apH of about 7.3 to about 7.5, including without limitation, any valueencompassed within these ranges but not explicitly recited herein. Inother embodiments the biomatrix scaffold, buffers, and/or media aremaintained at neutral pH. The temperature of the biomatrix scaffold(e.g., during and/or after preparation), buffers, and/or media can befrom about 0° C. to about 30° C., from about 5° C. to about 25° C., orfrom about 10° C. to about 20° C., including without limitation, anyvalue encompassed within these ranges but not explicitly recited herein.In some embodiments the temperature is maintained at about 20° C. Thetime for perfusing the biological tissue with any medium or buffer canbe from about 5 hours or less, about 3 hours or less, about 1 hour orless, about 30 minutes or less, or about 15 minutes or less. In someembodiments the step of perfusing the biological tissue with thedelipidating buffer is about 30 minutes or less. In some embodimentswhere acidic pHs are used, the salt concentrations for maintaining thecollagens and collagen-associated components insoluble can be different;the concentrations can be determined by the extant literature oncollagen chemistry by choosing salt concentrations that maintaininsolubility of the collagens.

Exemplary buffers include but are not limited to sodium chloride, sodiumlactate, sodium acetate, sodium phosphate, sodium citrate, sodiumborate, sodium gluconate, citrate buffers, bis\tris buffers, phosphatebuffers, potassium phosphate, citrate/dextrose, sodium bicarbonate,ammonium chloride, 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonicacid, tris(hydroxymethyl)methylamine,N-tris(hydroxymethyl)methylglycine,4-2-hydroxyethyl-1-piperazineethanesulfonic acid, and3-(N-morpholino)propanesulfonic acid.

In some embodiments the buffer of this invention (e.g., the buffer usedin step (c) described herein) can comprise a salt in a concentrationfrom about 2.0M or more. For example, in some embodiments the salt maybe in a concentration from about 2.0M to about 5.0M, from about 2.5M toabout 5.0M, from about 3.0M to about 4.5M, or from about 3.0M to about4.0M, including without limitation, any value encompassed within theseranges but not explicitly recited herein. For instance, in someembodiments the buffer utilized in the methods of the present inventioncan comprise a salt such as sodium chloride in a concentration fromabout 2.0M NaCl to about 4.5M NaCl. In other embodiments, such as thosefor adult livers, the buffer utilized can comprise from about 3.4M toabout 3.5M NaCl. In embodiments such as those for fetal liver, thebuffer utilized can comprise a salt such as sodium chloride in aconcentration from about 4.0M to about 4.50M. In some embodiments theperfusion of the biological tissue with a salt wash, such as that ofstep (c) of the exemplary methods described herein, is carried out untilthe perfusate (i.e., the fluid used for the perfusion, such as the fluidthat has been forced through the vasculature) is negative for proteinsby optical density (OD) at 280 nm.

Any of the media and/or buffers of the present invention may comprise aprotease inhibitor. Exemplary protease inhibitors are described above.In some embodiments the buffer such as that in step (c) of the exemplarymethods described herein comprises a protease inhibitor, such as soybeantrypsin inhibitor. In other embodiments the buffer of step (d) comprisesone or more protease inhibitors, such as soybean trypsin inhibitor.

The media and/or buffers of the present invention may comprise one ormore nucleases, which in some embodiments can be prepared in thestandard buffers recommended by the commercial suppliers of theseenzymes. For instance, in some embodiments the buffer of step (d)comprises one or more nucleases, such as but not limited to RNase andDNase. Perfusion with nucleases eliminates residues of nucleic acids. Inother embodiments the buffer of step (d) comprises RNase, DNase, and oneor more protease inhibitors. In some embodiments the perfusion of thebiological tissue with one or more nucleases is carried out until theperfusate (i.e., the fluid used for the perfusion, such as the fluidthat has been forced through the vasculature) is negative for nucleicacids by optical density (OD) at 260 nm. In some embodiments, thenucleases eliminate 75%, 80%, 85%, 90%, 95%, 98%, or 100% of nucleicacids in the biological tissue.

The second medium (e.g., final rinse medium) can be any medium thatensures that the collagens and bound factors (e.g., matrix components,growth factors, and cytokines) will remain insoluble, as describedabove. Exemplary final rinse media are described above in reference tothe first medium and are serum-free, at neutral pH, and with anosmolality of 250-350 mOsm/kg. For instance, in some embodiments thesecond medium comprises a basal medium. In some embodiments the secondmedium is a serum-free basal medium. In other embodiments, the secondmedium is a serum-free, hormonally defined medium (HDM) comprisinghormones, growth factors, lipids, and serum albumin and is tailored tothe need of the cells to be cultured. An exemplary second medium isKubota's medium (Kubota and Reid, PNAS 97:12132-12137, 2000), which isdesigned for hepatic stem cells, hepatoblasts and other progenitors. Incertain embodiments the second medium may or may not comprisesupplementation with serum or a factor derived from serum, such as butnot limited to human serum albumin. In some embodiments, rinsing thetissue with the second medium eliminates residues of the delipidatingbuffer and the nucleases. In other embodiments the wash with the secondmedium and/or any subsequent buffer or medium equilibrates the biomatrixscaffold with the medium or buffer. In some embodiments the first mediumand second medium can be the same and in some embodiments, the firstmedium and second medium can be different thereby producing a biomatrixscaffold from the biological tissue.

In some embodiments one or more of the media and/or buffers utilized inthe preparation of the biomatrix scaffold are free of (i.e., do notcontain a detectable amount of) one or more enzymes that degradeextracellular matrix components. In other embodiments all of the mediaand buffers utilized in the preparation of the biomatrix scaffold arefree of (i.e., do not contain a detectable amount of) one or moreenzymes that degrade extracellular matrix components. Exemplary enzymesinclude, but are not limited to collagenases; proteases; glycosidasessuch as heparinase, heparitinase, chondroitinase, and hyaluronidase; andelastases.

Sterilization of the biological tissue, homogenate and/or biomatrixscaffold of this invention can be accomplished by any method known inthe art, with the caveat that methods using a factor that can bind tothe biomatrix scaffold (e.g., ethylene oxide) should be avoided.Exemplary methods of sterilization include but are not limited to gammairradiation, radio frequency glow discharges (RFGD) plasmasterilization, electron beam sterilization, and super critical carbondioxide sterilization. In some embodiments sterilization of the tissue,homogenate and/or biomatrix scaffold is accomplished with gammairradiation at about 5,000 rads. If the scaffolds are to be usedimmediately for recellularization, and if sterile procedures were usedin the decellularization process (especially after the high saltextraction), then sterilization may not be required.

Storage of the biomatrix scaffold can be accomplished by any methodknown in the art. In some embodiments (e.g., when the scaffold is to beused intact), the biomatrix scaffold can be stored at about 4° C. and inother embodiments (e.g., when the scaffold is to be dispersed into sectthe biomatrix scaffold is frozen, for example, at about −80° C.

In some embodiments the biomatrix scaffold comprises, consists of, orconsists essentially of collagens, fibronectins, laminins,nidogen/entactin, elastin, proteogylcans, glycosaminoglycans and anycombination thereof, all being part of the biomatrix scaffold (e.g.,bound to the biomatrix scaffold). In some embodiments, the biomatrixscaffold lacks a detectable amount of a collagen, fibronectin, laminin,nidogen/entactin, elastin, proteogylcan, glycosaminoglycan and anycombination thereof.

The biomatrix scaffolds of the present invention have proven to bepotent differentiation substrata for cells and may be used for many celltypes, such as but not limited to any mature cell or for various stemcell populations. These include, e.g., embryonic stem (ES) cells,induced pluripotent stem (iPS) cells, germ layer stem cells (e.g.,definitive endodermal stem cells), determined stem cells (e.g., hepatic,lung, pancreatic or intestinal stem cells), human hepatic stem cells(hHpSCs), perinatal stem cells (e.g., amniotic fluid-derived stem cells(AFSCs)), mesenchymal stem cells (MSCs) such as from bone marrow or fromadipose tissue, committed progenitors, adult cells of any tissue type,diseased cells, tumor cells, mature cells, parenchymal cells, stellatecells, cholangiocytes, biliary tree cells such as those that are notcholangiocytes, hepatocytes, kidney cells, urothelial cells, mesenchymalcells, smooth or skeletal muscle cells, myocytes (muscle stem cells),fibroblasts, chondrocytes, adipocytes, fibromyoblasts, endothelialcells, ectodermal cells, including ductile and skin cells, neural cells,islet cells, cells present in the intestine, osteoblasts, other cellsforming bone or cartilage, and any combination thereof. These cells maybe normal or diseased.

In some embodiments the biomatrix scaffolds are used for biological,pharmaceutical, genetic, molecular, and/or virological studies of cells,whether freshly isolated from tissue or from lineage-restricted stemcells. In other embodiments the biomatrix scaffolds are used forimplantable, vascularized engineered organs, such as but not limited tothe liver. Other exemplary uses for the biomatrix scaffolds include, butare not limited to, protein manufacturing, drug toxicity testing, drugdevelopment, antibody screening, and/or virus production for vaccinepreparations of viruses. Virus production of lineage-dependent viruses(e.g., papilloma virus and hepatitis C) can be achieved by plating stemcell populations on a tissue-specific biomatrix scaffold and thenculturing in a medium that works in combination with the biomatrixscaffold to fully induce differentiation of the cells. The maturevirions will be produced when the cells fully mature. As long as thevirus itself does not affect cell viability, the mature cells infectedwith the virus can be maintained for at least eight weeks offering ameans of generating large amounts of virus with a stable culture system.

The biomatrix scaffolds can be used intact, such as but not limited touse for 2-D and/or 3-D cultures for cells. In some embodiments, thebiomatrix scaffolds can be used in combination with specific medium fordifferentiation in 2-D and/or 3-D cultures for cell lines, such as butnot limited to, normal or diseased cells from any maturational lineagestage from stem cells to late stage cells.

Alternatively, the biomatrix scaffolds can be frozen. These frozensections can be prepared and used as substrata. The biomatrix scaffoldscan be quickly frozen on dry ice and frozen sections prepared with aCryostat, placed onto culture apparati (e.g., dishes, flasks, cloth,transwells, etc.), sterilized and rehydrated in medium before seedingcells. In some embodiments, the frozen biomatrix scaffold of thisinvention can be sectioned.

In some embodiments a cell culture is produced, comprising: a) producinga biomatrix scaffold according to the methods of this invention; b)contacting the biomatrix scaffold of step (a) with cell culture mediumin a culture apparatus; and c) seeding the biomatrix scaffold of step(b) with cells, thereby producing a cell culture.

In some embodiments a cell culture is produced, comprising: a) producinga biomatrix scaffold of the present invention; b) freezing the biomatrixscaffold of step (a); c) preparing a frozen section from the biomatrixscaffold of step (b) as a cell culture substratum; d) contacting thecell culture substratum of step (c) with cell culture medium in aculture apparatus; and e) seeding the cell culture substratum of step(d) with cells, thereby producing a cell culture.

In other embodiments the biomatrix scaffolds can be ground to a powder.One method of grinding the biomatrix scaffold to a powder comprisesgrinding the biomatrix scaffold to a powder in a freezer mill attemperatures at or near liquid nitrogen temperatures. Other apparatusfor grinding at liquid nitrogen or equivalent temperatures (e.g.,freezing with dry ice) are known in the art. The powder can be broughtto room temperature at which it acquires the consistency of a paint thatcan be coated onto culture apparati using a sterilized paint brush orequivalent apparatus. The powder or the plates can be sterilized.

Thus, in some embodiments a cell culture is produced comprising: a)producing a biomatrix scaffold of the present invention; b) grinding thebiomatrix scaffold of step (a) to a powder; c) coating a cultureapparatus with the powder of step (b) to produce a cell culturesubstratum; d) contacting the cell culture substratum of (c) with cellculture medium in the culture apparatus; and e) seeding the cell culturesubstratum of (d) with cells, thereby producing a cell culture. In someembodiments of this method, the grinding of the biomatrix can be carriedout in a freezer mill (e.g., cryogenic grinding) at or near liquidnitrogen temperature.

In some embodiments before seeding the cells for the cell culture, aportion of the medium is added to the culture apparatus since the cellsmay attach within seconds. The cells, in some embodiments attach withinseconds to minutes for normal adult cells and within minutes to a fewhours for various types of stem cells. In some embodiments theattachment of the cells may depend on how the biomatrix scaffolds aredispersed for use in cultures. The cell medium can be any medium that issuitable for producing a cell culture. In some embodiments the cellculture medium comprises at least one constituent present ininterstitial fluid, wherein the osmolality of said medium is from about250 mOsm/kg to about 350 mOsm/kg, wherein said medium is serum free andwherein the pH is neutral. In other embodiments the cell culture mediumcan be a basal medium, such as but not limited to RPMI-1640, DME/F12,Ham's medium, Kubota's medium, etc.

The cell cultures produced with the biomatrix scaffolds, in someembodiments, comprise, consist essentially of or consist of, the sametype of cells as the cells of the biological tissue that was used tomake the biomatrix scaffold. Nonlimiting examples of the cells of thisinvention include embryonic stem (ES) cells, induced pluripotent stem(iPS) cells, determined stem cells, perinatal stem cells, amnioticfluid-derived stem cells (AFSCs), mesenchymal stem cells (MSCs) from anysource, committed progenitors or adult cells of any tissue type, maturecells, normal cells, diseased cells, tumor cells and any combinationthereof. Additional nonlimiting examples include liver cells,parenchymal cells, stellate cells, endothelial cells, hepatocytes,cholangiocytes, biliary tree cells that are not cholangiocytes andpancreatic cells.

In some embodiments, the primitive stem cells (whether ES, iPS, MSC orAFSC) will lineage-restrict the cells, at least partially, to the tissuetype used to make the biomatrix scaffold. Determined stem cells of agiven germ layer will lineage restrict to the tissue type used to makethe biomatrix scaffold when the scaffold is prepared from a tissuederived from that germ layer and may partially differentiate to theadult fate if on a scaffold from a tissue derived from a different germlayer. Thus, the ability of adult cells to fully differentiate may bedictated by the tissue type of the biomatrix scaffold. In parallel, thefate of stem cells may be partially or fully dictated by the tissue typeof the biomatrix scaffold or the fate of stems cells may be fullydictated by the tissue type of the biomatrix scaffold. In someembodiments, the cells of the cell culture are of a different type thanthe cells of the biological tissue used to make the biomatrix scaffold.As described in detail above, exemplary types of cells that may be usedin producing a cell culture include but are not limited to embryonicstem cells, induced pluripotent stem cells, mesenchymal stem cells,amniotic fluid derived stem cells, determined stem cells, mature cells,normal cells, diseased cells, tumor cells and any combination thereof.These cells may be from any biological tissue as described herein.

In some embodiments the biomatrix scaffolds induce slow growth or growtharrest correlated with differentiation of the normal cells, whether stemcells or mature cells. The mature cells, in some embodiments, becomefully differentiated within hours and remain stably differentiated forat least eight weeks thereafter. In some embodiments adult cells (i.e.,fully mature cells) attach to the scaffolds within minutes and retaintheir full differentiation thereafter for more than eight weeks. Stemcells, in some embodiments, undergo a few divisions and then go intogrowth arrest and fully differentiate. The stem cells remain stably ingrowth arrest, viable and fully differentiated for at least eight weeks.In some embodiments, stem cells seeded onto biomatrix scaffolds go intogrowth arrest or slowed growth, lose stem cell markers and differentiateto mature, functional cells in approximately one week, retaining stablephenotypes and viabilities for at least eight weeks or more (e.g., 40%,50%, 60%, 70%, 80%, 90%, 95%, or 100% viability for an extended periodof time (e.g., at least one week, at least two weeks, at least threeweeks, at least four weeks, at least five weeks, at least six weeks, atleast seven weeks, at least eight weeks, at least nine weeks, at leastten weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, atleast 14 weeks, at least 15 weeks, at least 16 weeks, at least onemonth, at least two months, at least three months, at least four months,at least five months, at least six months, at least seven months, atleast eight months, at least nine months, at least ten months, at least11 months, at least one year, etc.)

In other embodiments the biomatrix scaffold is used to differentiateembryonic stem (ES) cells and/or induce pluripotent stem (IPS) cells toa specific fate. For instance, in some embodiments a tissue-specificbiomatrix scaffold is used to facilitate differentiating embryonic stemcells or induced pluripotent cells towards a specific fate.

In certain embodiments of the present invention the biomatrix scaffoldis used to differentiate amniotic fluid-derived stem cells (AFSCs) ormesenchymal stem cells (MSCs) from bone marrow or from adipose tissue orfrom any fetal or postnatal tissue or any determined stem cells (e.g.,lung, intestine, biliary tree, kidney, skin, heart, etc.) towards aspecific adult fate. In some embodiments the biomatrix scaffolds of thepresent invention are used to enhance and accelerate differentiation ofstem cells to mature cells by producing a cell culture.

In other embodiments the present invention provides a method ofenhancing and/or accelerating differentiation of stem cells and/orprogenitors to mature cells, comprising producing a cell cultureaccording to the methods of this invention, wherein the cells are stemcells and the cell culture medium is formulated for mature cells,thereby enhancing and/or accelerating differentiation of stem cellsand/or progenitors to mature cells. The cell culture medium can be anymedium that is formulated for mature cells. The constituents in themedium are distinct for each cell type. Differentiation means that theconditions cause the cells to mature to adult cell types that produceadult specific gene products. Cells employed in these methods can beadult cells of any type or stem cells or progenitors, nonlimitingexamples of which include embryonic stem cells, induced pluripotent stemcells, germ layer stem cells, determined stem cells, perinatal stemcells, amniotic fluid-derived stem cells, mesenchymal stem cells,transit amplifying cells or committed progenitors of any tissue type.

Cells seeded onto the biomatrix scaffolds (e.g., intact biomatrixscaffolds, sections of scaffolds or powdered biomatrix scaffolds mixedinto/onto other implantable materials) can be transplanted into animalsor humans as a method of grafting cells in vivo. In some embodiments amethod of delivering cells to a subject is provided comprisingcontacting the subject with the biomatrix scaffold of the presentinvention, where the biomatrix scaffold comprises cells. In otherembodiments a method of delivering cells to a subject is provided thatcomprises seeding the biomatrix scaffold of the present invention withcells and then transplanting the biomatrix scaffold seeded with thecells into the subject. In some embodiments, a biomatrix scaffold thathas not been seeded with any cells can be transplanted into a subject.

In some embodiments, the biomatrix scaffold can be used as a graft thatcan be used to regenerate the tissue or organ in the subject.

The biomatrix scaffolds of the present invention may be used forestablishing bioartificial organs, which may be useful for analyticaland/or clinical programs. The biomatrix scaffolds may also be used foridentifying specific gene products or facets of disease states. In someembodiments the biomatrix scaffolds are prepared from tissues of mutantanimals and subsequently used to define relevant factor(s) associatedwith the mutation(s). In other embodiments the biomatrix scaffolds areprepared from diseased tissues and used to define changes in the matrixrelevant to the disease.

Additional nonlimiting examples of uses of the biomatrix scaffolds ofthis invention include: 1) use of the scaffold for culturing malignantcells to define metastatic potential (the ability of tumor cells to formgrowing colonies of cells on a given type of biomatrix scaffold ispredictive of the ability of the cells to metastasize to the tissue fromwhich that scaffold was prepared; 2) putting grafts of tissue on thescaffold to be used for transplantation into a subject); 3) productionof organoids formed by recellularization of scaffolds to be used asassist devices, such as, for example, a liver organoid that is thenconnected to a subject with liver failure; 4) use of the scaffold forprotein manufacturing (cells on the scaffold produce a factor that canbe isolated from the medium and/or from the cells and then purified; and5) use of the scaffold for production of lineage dependent virnses;e.g., for production of viruses that require differentiated cells toyield sufficient particles for use as a vaccine.

Thus, the present invention provides a method of identifying themetastatic potential of tumor cells in a tissue type, comprising a)producing a biomatrix scaffold according to the methods of thisinvention; b) contacting the biomatrix scaffold of (a) with cell culturemedium in a culture apparatus; c) seeding the biomatrix scaffold of (b)with tumor cells; d) maintaining the biomatrix scaffold of (c) underculture conditions; and e) monitoring growth of the tumor cells on thebiomatrix scaffold of (d), wherein growth of tumor cells on thebiomatrix scaffold identifies that the tumor cells can colonize in vivothe type of tissue from which the biomatrix scaffold was produced,thereby identifying the metastatic potential of the tumor cells in thetissue type.

Also provided herein is a method of identifying a tumor cell asresponsive to an anti-tumor treatment, comprising: a) producing abiomatrix scaffold according to the methods of this invention; b)contacting the biomatrix scaffold of (a) with cell culture medium in aculture apparatus; c) seeding the biomatrix scaffold of (b) with tumorcells; d) maintaining the biomatrix scaffold of (c) under cultureconditions; e) applying the anti-tumor treatment to the tumor cells onthe biomatrix scaffold; and f) monitoring growth of the tumor cells onthe biomatrix scaffold of (e), wherein lack of growth of tumor cellsand/or death of tumor cells on the biomatrix scaffold of (e) identifiesthe tumor cells as responsive to the anti-tumor treatment. Nonlimitingexamples of anti-tumor treatment include chemotherapeutic agents,antibodies, radiation therapy, immunotherapy, hormonal therapy etc., aswould be well known in the art. In some embodiments, tumor cells from asubject can be seeded unto different biomatrix scaffolds of thisinvention and exposed to respective anti-tumor treatments. Pursuant tothe results of these respective analysis of different anti-tumortreatments, an anti-tumor treatment that is effective against thesubject's tumor cells can be selected and that anti-tumor treatment canbe administered to the subject to treat the subject's tumor.

In further embodiments, the present invention provides a method ofproducing a tumor graft for transplantation into a host animal,comprising: a) producing a biomatrix scaffold according to the methodsof this invention; b) contacting the biomatrix scaffold of (a) with cellculture medium in a culture apparatus; c) seeding the biomatrix scaffoldof (b) with tumor cells; d) maintaining the biomatrix scaffold of (c)under culture conditions; and e) establishing a population of the tumorcells on the biomatrix scaffold of (d), thereby producing a tumor graftfor transplantation into the host animal. In some embodiments, thismethod can further comprise the step of transplanting the tumor graftinto the host animal. In various embodiments, the tumor graft can besyngeneic, allogeneic or xenogeneic to the host animal.

Also provided herein is a method of producing virus particles of alineage dependent virus, comprising: a) producing a biomatrix scaffoldaccording to the methods of this invention; b) contacting the biomatrixscaffold of (a) with cell culture medium in a culture apparatus; c)seeding the biomatrix scaffold of (b) with cells of a type and lineagestage that can be infected with the lineage dependent virus; d)infecting the cells of (c) with the lineage dependent virus; e)maintaining the infected cells on the biomatrix scaffold under cultureconditions; and f) collecting virus particles produced in the infectedcells, thereby producing virus particles of the lineage dependent virus.

Nonlimiting examples of a lineage dependent virus of this inventioninclude hepatitis C virus, hepatitis B virus, norovirus human papillomavirus and any other virus now known or later identified to be lineagedependent. By lineage dependent is meant that the cell in which thevirus is present must mature or differentiate to a particular stagebefore the virus can successfully replicate in the cell and producevirus particles, as is known in the art.

Furthermore, the present invention provides a method of producing anorganoid formed by recellularization of a biomatrix scaffold,comprising: a) producing a biomatrix scaffold according to the methodsof this invention; b) contacting the biomatrix scaffold of (a) with cellculture medium in a culture apparatus; c) seeding the biomatrix scaffoldof (b) with cells of the same tissue type as the biological tissue usedto prepare the biomatrix scaffold; and d) maintaining the cells on thebiomatrix scaffold under culture conditions, whereby organoids form fromthe cells, thereby producing an organoid formed by recellularization ofthe biomatrix scaffold. This method can further comprise the step ofcontacting the organoid produced by steps (a) through (d) with asubject, for use as an assist device, as is known in the art. Any celltype that can be used to produce the biomatrix scaffold of thisinvention can be used in this method. In some embodiments, the cells areliver cells.

The present invention additionally provides a method of producing aprotein of interest in cells cultured on a biomatrix scaffold,comprising: a) producing a biomatrix scaffold according to the methodsof this invention; b) contacting the biomatrix scaffold of (a) with cellculture medium in a culture apparatus; c) seeding the biomatrix scaffoldof (b) with cells that produce the protein of interest; d) maintainingthe cells of (c) on the biomatrix scaffold under culture conditions; ande) collecting the protein of interest produced by the cells of (d),thereby producing a protein of interest in cells cultured on a biomatrixscaffold. This method can comprise the further step of purifying theprotein of interest collected in step (f). The protein of interest ofthis invention can be any protein produced by a cell, either from anendogenous gene and/or as a recombinant protein, in an amount that canbe collected from the cells in culture and/or the culture medium.Numerous examples of such proteins of interest are known in the art.

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLES Example 1. Lineage Restriction of Human Hepatic Stem Cells toMature Fates is Made Efficient by Tissue-Specific Biomatrix Scaffolds

Abstract.

Current protocols for differentiation of stem cells make use of multipletreatments of soluble signals and/or matrix factors and result typicallyin partial differentiation to mature cells with under- or overexpressionof adult tissue-specific genes. In the present invention, a strategy wasdeveloped for rapid and efficient differentiation of stem cells usingsubstrata of biomatrix scaffolds, tissue-specific extracts enriched inextracellular matrix, and associated growth factors and cytokines, incombination with a serum-free, hormonally defined medium (HDM) tailoredfor the adult cell type of interest. The studies described hereindemonstrate the efficacy of the biomatrix scaffolds of this invention indifferentiating human hepatic stem cells (hHpSCs) to mature fates and inmaintaining mature parenchymal cells as fully functional for longperiods of time. Biomatrix scaffolds were prepared by a novel four-stepperfusion decellularization protocol using conditions designed to keepall collagen types insoluble. The scaffolds maintained native histology,patent vasculatures and approximately 1% of the tissue proteins but >95%of its collagens, most of the tissue's collagen-associated matrixcomponents, and physiological levels of matrix bound growth factors andcytokines. Collagens increased from almost undetectable levels to >15%of the scaffold's proteins with the remainder including laminins,fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-boundcytokines and growth factors in patterns that correlated with histology.Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffoldsand in an HDM tailored for adult liver cells, lost stem cell markers anddifferentiated to mature, functional parenchymal cells in approximatelyone week, remaining viable and with stable mature cell phenotypes formore than eight weeks. Thus, the biomatrix scaffolds of this inventioncan be used for biological and pharmaceutical studies oflineage-restricted stem cells, for maintenance of mature cells, and forimplantable, vascularized engineered tissues or organs.

Procedures for Decellularization.

After anesthesia with ketamine-xylazine, the rat abdominal cavity wasopened and a sleevelet with a cannula was inserted into the portal veinto perfuse the entire liver. (1) Perfusion is done with RPMI 1640 for 10mins; followed by (2) delipidation with a lipase (e.g., 20-50 units ofphospholipase A2-PLA2) combined with a gentle detergent such as 1%sodium deoxycholate (SDC) for about 30-60 mins until the tissue becomestransparent and the effusion becomes clear; (3) perfusion with high saltwashes (for fetal livers: 4.5M NaCl. and for adult livers 3.4M-3.5MNaCl.) is done until the perfusate is negative for proteins by opticaldensity (OD) at 280 nm; (4) perfusion with nucleases (DNase, RNase) inRPMI 1640 until the perfusate is negative for nucleic acids by OD 260;and (5) final rinse with RPMI 1640 for 2 hours or more.

The biomatrix scaffolds are quickly frozen on dry ice and frozensections prepared with a Cryostat, placed onto 24-well cell cultureplates, sterilized by gamma irradiation (5000 rads) and rehydrated inmedium (KM) for 30 min before seeding cells. The sections of biomatrixscaffolds covered 95% of well surface in the 24-well plate.

An alternative method for distributing the biomatrix scaffolds ontoculture dishes consisted of pulverizing it to a powder using a freezermill filled with liquid nitrogen. The pulverized powder, when brought toroom temperature, acquires the consistency of paint, and can be coatedonto any surface, such as dishes, slides, cloth, filters or othersurfaces used for attaching cells and/or cell culture. Pulverizing thescaffolds eliminates the gradients of matrix components and signals, butthe mix of components present still elicits potent differentiationeffects. The scaffolds also can be used intact and reseeded with cellsin preparation of engineered organs for transplantation in vivo or for3-D cultures.

Alternate methods were developed for use with porcine and bovine livers.Pig and bovine livers were obtained from a USDA certified meatprocessing facility (CT). See Example 3 for an outline of arepresentative protocol. Each liver was USDA inspected and received theUSDA stamp prior to leaving the facility. Livers were transported inESP-Gro medium (Gigacyte, Branford, Conn.; catalog #1101-250). Liversreceived in the laboratory were weighed, photo-documented and preparedfor perfusion. After grinding, the mixture was thawed and diluted to amedia:biomatrix ratio of 1:48. This biomatrix slurry was then used forcoating plates. After drying, the biomatrix was washed three times andthen cells were applied. Adult liver cells attached within 10 minutes tothe plates. Stem/progenitors can take longer (a few hours). However, forboth stem/progenitors and adult liver cells, essentially 100% of theviable cells attach.

Media and Solutions.

All media were sterile-filtered (0.22-μm filter) and kept in the dark at4° C. before use. To keep collagens stable in the biomatrix, the pH ofthe perfusion media for biomatrix scaffold preparation was kept at7.5-8.0. RPMI-1640 (Gibco/Invitrogen, Carlsbad, Calif.) was used as thebasal medium for preparation of biomatrix scaffolds and for hepatocyteor hepatic stem cell cultures. All reagents except those noted wereobtained from Sigma (St. Louis, Mo.).

Perfusion Media for Biomatrix Scaffold Preparation.

(1). Perfusion wash and perfusion rinse: serum-free basal medium (e.g.,RPMI-1640);(2). Perfusion with detergent: 36 units/L PLA2 plus 1% SDC;(3). Perfusion with high salt: 3.4M NaCl. with 0.1 mg/ml Soybean trypsininhibitor;(4). Perfusion with nucleases: 5 mg/100 ml RNase, 1 mg/100 ml DNase and0.1 mg/ml soybean trypsin inhibitor (e.g., prepared in RPMI 1640).

Kuhota's medium;

KM was designed originally for hepatoblasts⁴⁷ and now has been foundeffective for human hepatic progenitors⁴⁸ and for other endodermalprogenitors including ones from biliary tree (Wang et al. “Multipotentstem/progenitor cells in human biliary tree give rise to hepatocytes,cholangiocytes and pancreatic islets” Hepatology, 2011, in press) andpancreas (Wang, Y and Reid L, unpublished data). It consists of anybasal medium (here being RPMI 1640) with no copper, low calcium (0.3mM), 10-9 M Selenium, 0.1% BSA, 4.5 mM Nicotinamide, 0.1 nM Zinc Sulfateheptahydrate (from Specpure, Johnson Matthew Chemicals, Royston,England), 10-⁸M hydrocortisone, 5 μg/ml transferrin/Fe, 5 μg/ml insulin,10 μg/ml high density lipoprotein, and a mixture of free fatty acidsthat are added bound to purified human serum albumin.

To differentiate cells to an adult fate, a serum-free, hormonallydefined medium (HDM) tailored to the adult cell type desired can beused. For example, we used an HDM for the adult liver fate consisting ofKM supplemented further with calcium to achieve a 0.6 mM concentration,10-¹²M copper, 1 nM tri-iodothyronine (T3), 7 ng/ml glucagon, 20 ng/mlof FGF, 2 g/L galactose, 10 ng/ml Oncostatin M (OSM), 10 ng/ml epidermalgrowth factor (EGF), 20 ng/ml hepatocyte growth factor (HGF), and 10-⁸Mhydrocortisone.

The cells were seeded in this HDM serum-free if plating on thescaffolds; in circumstances in which enzymes were used for processingcells or tissues, then we supplemented the HDM with 5% FBS (HyClone,Waltham, Mass.) for a few hours and then switched to serum-free HDMthereafter. In parallel, control experiments, cultures were kept in theHDM with 5% FBS throughout, but we found that the presence of serumcaused cells to lose differentiated functions with time. The solublefactors requirements are less than normal for cultures on othersubstrata given that so many of the factors are bound to the biomatrixscaffolds. The soluble factors requirements are less than normal forcultures on other substrata given that so many of the factors are boundto the biomatrix scaffolds.

Characterization of Intact Vascular Trees in the Liver BiomatrixScaffolds.

The branching and ramifying matrix remnants of the vasculature includingthe network of capillaries in the rat liver biomatrix scaffold have beenvisualized by light and fluorescence microscopy, respectively.Rhodamine-labeled 250 kDa dextran particles were injected into the liverbiomatrix scaffold through the remnant of the portal vein to check theintegrity of the matrix remnants of the vasculature system in thebiomatrix scaffolds. A movie was prepared using a Leica MZ16FAfluorescence dissecting microscope (motorized).

Human Fetal Liver Processing.

Fetal liver tissues were provided by an accredited agency (AdvancedBiological Resources, San Francisco, Calif.) from fetuses between 16-20weeks gestational age obtained by elective pregnancy terminations. Theresearch protocol was reviewed and approved by the Institutional ReviewBoard for Human Research Studies at the University of North Carolina atChapel Hill. Suspensions of fetal human liver cells were prepared asdescribed previously^(48,49). Briefly, processing was conducted in RPMI1640 supplemented with 0.1% bovine serum albumin, 1 nM selenium andantibiotics. Enzymatic processing buffer contained 300 U/ml type IVcollagenase and 0.3 mg/ml deoxyribonuclease at 32° C. with frequentagitation for 15-20 min. Enriched suspensions were pressed through a 75gauge mesh and spun at 1200 RPM for 5 min before resuspension. Estimatedcell viability by trypan blue exclusion was routinely higher than 95%.

Enrichment of hHpSCs and Culture on Biomatrix Scaffolds.

We used two methods for the hHpSCs purification or enrichment:

1) Culture selection. Approximately 3×10⁵ cells were plated on a 10 cmtissue culture dish and in KM. Medium was changed every 3 days. Coloniesformed within 5-7 days and were observed for up to 3 months. We pickedcolonies by hand after 14-18 days using an inverted microscope(lX-FLAIII; Olympus, Japan and Melville, N.Y.).

2) Magnetic immunoselection of multipotent hepatic progenitorsubpopulations (hHpSCs and hHBs) was achieved by selection of cellspositive for epithelial cell adhesion molecule (EpCAM, CD326) usingmagnetic bead immunoselection technologies with the Miltenyi BiotechMACS system (Bergisch Gladbach, Germany) following the manufacturer'sinstructions⁵⁰. Briefly, the dissociated cells were incubated with EpCAMantibody bound to magnetic microbeads for 30 min at 4 C, and wereseparated using a magnetic column separation system from Miltenyifollowing the manufacturer's recommended procedures.

Cultures were seeded with either 250 hHpSC colonies, or 5×10⁵ enrichedhHpSCs or 2.5×10⁵ primary adult hepatocytes. Medium was replaced dailyand collected medium was stored at −20° C. for further analysis. Cellscultured on Collagen type I coating 24-well plates served as control.

Adult Rat Jiepatocyte Isolation.

Freshly isolated suspensions of rat hepatocytes were obtained from3-month old adult male Lewis rats (Charles River Laboratories,Wilmington, Mass.) weighing 200-250 g. An improved two-step perfusionmethod as previously described⁴⁹ was used for rat hepatocyte isolationand purification. The liver was perfused for 10-15 minutes with acalcium-free buffer containing EGTA and then collagenase in acalcium-containing buffer for 10-15 minutes. The liver was thenmechanically dissociated by pressing the digested liver through cheesecloth and then sequentially filtering the cell suspension through sievesof narrowing mesh size. The cells were washed twice and then pelleted at50 g. Viability was defined by counting the cells after trypan bluestaining. Routinely, 200-300 million cells per rat were isolated with89-96% viability and >99% purity.

Human Adult Liver Cell Isolation and Culture.

Fresh human liver cell suspensions were obtained from CellzDirect. (nowa part of Invitrogen, RTP, NC). Suspensions were processed perCellzDirect methods then resuspended in HeptoMAIN medium (Catalog#1103-250; GigaCyte, Branford, Conn.) plated at 1.88×105 cells/cm2 intomulti-well plates coated with liver biomatrix scaffolds or onto type Icollagen (1 μg/ml; Meridian Catalog #A33704H).

Collagen chemistry analysis. The amount of collagen in biomatrixscaffolds was evaluated based on the hydroxyproline (hyp) content.Samples of whole livers and of biomatrix scaffolds were pulverized,washed and lyophilized. Aliquots were then hydrolyzed and subjected toamino acid analysis⁵¹, and the collagen content per total protein wasestimated based on the hyp value of 300 residues/collagen.

Quantitative Analysis of DNA and RNA Content.

To assess total DNA remaining in the decellularized liver biomatrix,both fresh rat liver tissue and decellularized biomatrix were weighed,cut and digested with Proteinase K and total cellular DNA wasisolated⁵². To assess total RNA remaining in the decellularized liverbiomatrix, both fresh rat liver tissue and decellularized rat liverbiomatrix were weighed and then homogenized in TRizol solution(Invitrogen), and total cellular RNA was isolated.

Growth Factor Assays.

Samples of rat livers, rat liver biomatrix scaffolds, human bile ducttissue and human bile duct biomatrix scaffolds (two samples each) weresent to RayBiotech, Inc (Norcross, Ga.) for analysis of growth factors.The samples were homogenized, prepared as lysates, and then assayed with1 mg/ml protein, yielding fluorescence, defined in fluorescent intensityunits (FIUs). Semi-quantitative growth factor assays were done using theRayBio® Human Growth Factor Arrays, G Series 1. The FIUs were reduced bythat from negative controls for non-specific binding and normalized toprotein concentration. The data from the duplicates were averaged. Fourarrays were used enabling the survey for 40 growth factors. Although theassay was developed for human growth factors, there is sufficientoverlap in cross-reaction to rat growth factors to permit use for boththe rat and human samples.

Transmission and Scanning Electron Microscopy (TEM and SEM).

For TEM, the biomatrix scaffolds were rinsed with phosphate bufferedsaline (PBS) and fixed in 3% glutaraldehyde/0.1 sodium cacodylate, pH7.4 overnight. Following three rinses with sodium cacodylate buffer, thebiomatrix scaffolds were postfixed for 1 hour in 1% osmium tetroxide/0.1sodium cacodylate buffer. After rinsing in deionized water, it wasdehydrated and embedded in Polybed 812 epoxy resin (Polysciences, Niles,Ill.). The biomatrix scaffolds were sectioned perpendicular to thesubstrate at 70 nm using a diamond knife. Ultrathin sections werecollected on 200 mesh copper grids and stained with 4% aqueous uranylacetate for 15 minutes, followed by Reynolds' lead citrate for 7minutes. Samples were viewed using a LEO EM910 transmission electronmicroscope operating at 80 kV (LEO Electron Microscopy, Oberkochen,Germany). Digital images were acquired using a Gatan Orius SC1000 CCDDigital Camera and Digital Micrograph 3.11.0 (Gatan, Pleasanton,Calif.).

For SEM, after fixation and rinses, the biomatrix scaffolds weredehydrated and transferred in 100% ethanol to the Balzers CPD-020critical point dryer (Bal-Tee AG, Balzers, Switzerland), and dried usingcarbon dioxide as the transition solvent. The matrix was mounted onaluminum specimen supports with carbon adhesive tabs, and coated with a10 nm thickness of gold-palladium metal (60:40 alloy) using a Hummer Xsputter coater (Anatech, Worcester Mass.). Samples were examined using aZeiss Supra 55 FESEM at an acceleration voltage of 5 kV and digitalimages were acquired using Zeiss SmartSEM software (Carl Zeiss SMT,Germany and Thornwood, N.Y.).

Immunocytochemistry and Immunohistology.

For the fluorescent staining of cultured cells on biomatrix scaffolds,cells were fixed with 4% paraformaldehyde (PFA) for 20 min at roomtemperature, rinsed with HBSS, blocking with 10% goat serum in HBSS for2 h, and rinsed. Fixed cells were incubated with primary antibodies at4° C. overnight, washed, incubated for 1 h with labeled isotype-specificsecondary antibodies, washed, counterstained with4′,6-diamidino-2-phenylindole (DAPI) for visualization of cell nucleiand viewed using a Leica DMIRB inverted microscope (Leica, Houston,Tex.).

For immunohistochemistry, the biomatrix scaffolds were fixed in 4% PFAovernight and stored in 70% ethanol. They were embedded in paraffin andcut into 5-μm sections. Sections were deparaffinized, and the antigenswere retrieved. Endogenous peroxidases were blocked by incubation for 30min in 0.3% H202 solution. After blocking with 10% horse serum, primaryantibody was applied at 4° C. overnight; secondary antibody and ABCstaining were performed using the RTU Vectastain kit (VectorLaboratories, Burlingame, Calif.). Vector Nova RED was used assubstrate. Sections were dehydrated, fixed and embedded in EukittMounting Media (Electron Microscopy Sciences, Hatfield, Pa.), and wereanalyzed using an inverted microscope. Antibodies used for liversections and for cultures are listed in Table 4.

Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis.

The hHpSCs are cultured on cell culture plates, and the colonies weretransferred onto biomatrix scaffold. After further culture for 7 days,the colonies were lysed for RT-PCR. Total RNA was extracted using anRNeasy Plus Mini Kit (Qiagen GmbH, Valencia Calif.) as per themanufacturer's instructions. Reverse transcription was carried out withthe SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen,Carlsbad, Calif.). HotStarTaq Master Mix Kit (Qiagen) was used for PCR.PCR primers were listed in Table 5.

LIVE/DEAD Assay and Cell Viability Assay.

A LIVE/DEAD viability assay kit (Molecular Probes/Invitrogen, Carlsbad,Calif.) was used for the adhesion and proliferation assays. The hHpSCsor hepatocytes were incubated with two probes, calcein-AM (Live, lightgrey) and ethidium homodimer-1 (EtdD-1, Dead), for intracellularesterase activity and plasma membrane integrity, respectively. Specimenswere observed under a fluorescence Olympus SZX12 stereomicroscope(OLYMPUS, Japan and Melville, N.Y.). A resazurin cell viability assaykit (Biotium, Hayward, Calif.) was used following the manufacturer'smanual. Briefly, 10% of resazurin solution was added into culture mediumand incubated at 37° C. overnight. Absorbance OD₅₇₀-OD₆₀₀ was obtainedusing a Biotek Synergy HT multi-detection microplate reader (Winooski,Vt.) and the viability curve plotted. All experiments were carried outthree times using a minimum of three samples per experimental condition.

Hepatic Specific Functional Assays.

CYP450 3A4 activity was detected using a P450-Glo™ Screening System(Promega, Madison, Wis.). Briefly, the cultured cells were incubatedwith medium containing the luminogenic CYP3A4 substrate, luciferin-PPXEfor CYP, for 4 hours at 37° C. The luciferin detection and analysis wasperformed per the manufacturer's instructions with a Wallace Victor2Multilabel Counter (now part of Perkins/Elmer in Waltham, Mass.).Quantitative albumin secretion was done using a human albumin ELISAquantitation kit (Bethyl Laboratories, Montgomery, Tex.). For ureasynthesis assays, the cells were incubated with 2 mM ammonium for 24hours and the supernatant was collected and assayed with the Quantichromurea assay kit (Bioassay Systems, Hayward, Calif.). The supernatant fromone sample for each culture condition was assayed in triplicate and theexperiment was repeated 3 times.

Statistical Analysis.

Experiments were repeated at least 2-3 times with duplicate ortriplicate samples for each condition. Data from representativeexperiments are presented, whereas similar trends were seen in multipletrials. All error bars represent S.E.M.

Biomatrix Scaffolds are Prepared with a Novel Four-Step Protocol.

Biomatrix scaffolds were prepared using a protocol comprised ofdelipidation followed by high salt extractions and using perfusionmethods (FIG. 1). A detailed presentation of the protocol is given inthe methods. This is achieved by a novel four step protocol: 1) gentledelipidation; 2) washes with buffers with salt concentrations from about2.0M to about 5.0M (e.g., 2.0M-2.5M, 2.6M-3.0M; 3.1M-3.5M, 3.6M-4.0M,4.1M-4.5M; 4.6M-5.0M), salt concentrations known to maintain thecollagens in an insoluble state²³ (the exact concentration and the pH ofthe buffers is dictated by 2.5 the collagen types in the tissue),concentrations known to maintain collagens in an insoluble state²³; 3)nuclease treatment to eliminate residual nucleic acids; and 4) rinseswith a basal medium to eliminate the detergent, salt and nucleaseresidues as well as to equilibrate the matrix components with the medium(FIG. 1A).

The choices of the rinse media or the buffers for the nucleases can beany of a number of options as long as the salt concentration and ionicstrength are such as to maintain the matrix components in an insolublestate. The choice of the delipidation method is critical to be effectiveand yet gentle. We chose a combination of sodium deoxycholate (SDC) andPhospholipase A2 (PLA2) to rapidly degrade the phosphoglyceride locatedon the cytoplasm membrane and mitochondrial membrane into lysolecithin,a powerful surfactant, which can induce necrosis and cytolysis. Thereactive formula is shown in FIG. 8. We avoided harsh detergents, suchas sodium dodecyl sulfate (SDS) or Triton-X 100, which might dissolvesome matrix components such as the glycosaminoglycans (See review byGilbert et al. “Decellularization of tissues and organs” Biomaterials27:3675-3683 (2006)).

We avoided prolonged exposure of the scaffolds to the enzymes from thedisrupted cells during delipidation and the high salt washes, becausethey can greatly decrease the content of elastin and the content ofglycosaminoglycans (GAGs) such as heparan sulfates (HS), chondroitinsulfates (CS), dermatan sulfates (DS) and heparins (HP), which are sitesat which cytokines and growth factors bind²⁴. We used soybean trypsininhibitor and careful control of the pH (7.5-8.0), temperature (20° C.),and time (30-60 mins) to limit the activity of the proteases derivedfrom disrupted cells.

We perfused the whole tissue through relevant vasculature (e.g., portalvein in the liver), enabling us to rapidly isolate (within a few hours)a biomatrix scaffold with minimal loss of matrix components. Therapidity of the isolation is due to the initial step with detergent thatdelipidates the tissue within approximately 30-60 minutes (not hours ordays as in protocols used by others). The resulting biomatrix scaffoldsare translucent or white (FIG. 1). Moreover, using this perfusionmethod, we maintained the primary vasculature channels, portal andhepatic vein and most of the vascular branches in the liver, whichincreased the decellularization efficiency. Fluorescentrhodamine-labeled dextran particles perfused through the biomatrixscaffolds remained within the remnants of the vasculature demonstratingthat they are patent (FIG. 1E) There is a progressive flow of the dyefrom large vessels to the fine blood vessel branches along the channelswithout leakage. This fact will be helpful in revascularization of thescaffolds as a means of preparing engineered tissues for eitherthree-dimensional culture and/or for implantation ex vivo.

When sectioned, the scaffolds retain the histological structure of theoriginal tissue, including the recognizable remnants of majorhistological entities such as the blood vessels, bile ducts, andGlisson's capsule (GC) (FIG. 1). Compare FIGS. 1B1 and 1D1, in which asection of the liver tissue is contrasted with that of a biomatrixscaffold. The matrix remnants of the muralia of parenchymal cellsconsisted of a lace-like network (FIGS. 1D2-1D3).

Collagen, Collagen Associate Proteins and Bound Cytokines are Maintainedin the Biomatrix Scaffolds.

The amount of collagen in biomatrix scaffolds was evaluated by aminoacid analysis by methods used previously²⁵. Because hydroxyproline (Hyp)is unique to collagens and collagenous proteins, the collagencomposition relative to total protein was expressed as residues of Hypper 1,000 amino acids. The results demonstrated that the collagencontent increased from almost undetectable levels, i.e., less than 0.2residues of hydroxyproline (Hyp)/1,000 in liver, to 13 residues ofHyp/1,000 in biomatrix scaffolds. This indicates that delipidation andthe high salt washes, described above, did not remove collagens, leavingalmost all of the collagens in the biomatrix scaffolds. Detection ofsignificant levels of hydroxylysine (Hyl), another collagen-associatedamino acid, and higher levels of glycine (Gly) in biomatrix scaffoldsupport our conclusion that collagen is markedly enriched in biomatrixscaffolds (FIGS. 2A, 9 and Table 2).

Using immunohistochemical and ultrastructural studies, we were able toidentify in the scaffolds all known forms of collagens found in liver insitu including fibrillar collagens (collagen types I, III and V, 10-30nm in diameter for fibrils and 500-3,000 nm for assembled fibers) andbeaded filaments (possibly type VI). Those fibers and filaments arepresent in the subcapsular connective tissue layer lying beneath themesothelial layer. Although typical structures of basement membraneswere not found along the sinusoids from portal triads to central veins,we found that collagen type IV and some bound small fibrils formnet-like, porous 3D lattices, serving as scaffolding for the parenchymalcells (FIG. 2). Collagen type I bundles can be viewed as the principalstructure of the scaffolds to which other collagen types, glycoproteins,and proteoglycans are attached. In the space of Disse we found smallbundles of collagen type I and fibers of collagen types III and VI aswell as some type V, which is more abundant near portal triads andcentral veins. Representative immunohistochemistry data are presented inFIG. 3B, and a summary of matrix components and their location in normalliver tissue versus those in the biomatrix scaffolds are listed in FIG.4D. Early studies in the development of the protocols for biomatrixscaffold preparation indicated that the bulk of the cytoskeletalcomponents are lost in the washes. Still, we assessed the scaffolds byimmunohistochemistry and found no evidence for tubulin, desmin or actin,trace amounts of cytokeratins 18 and 19, and low levels of vimentinscattered throughout the scaffolds. The matrix associated with the bileducts and portions of the hepatic vascular systems (arterial and venousvessels) consists of typical basement membrane structures and so isquite distinctive from the thin layers of the matrix associated with thevascular structures found in the IO sinusoids. Laminin,entactin/nidogen, perlecan and collagen type IV are found in the portaltriad, whereas only perlecan and some collagen type IV are found in theSpace of Disse. Enormous amounts of hydrophobic, wavy elastin arepresent; it crosslinks together and forms sheets and fibers restrictedprimarily to the subcapsular connective tissue, portal regions, andarterial walls. Fibronectins are ubiquitous and prevalent throughout thehepatic matrix and are especially abundant in the Space of Disse, wherethey form either fine filaments or granular deposits (FIGS. 2 and 3).

Immunohistochemistry indicates that the known proteoglycans in thetissue are preserved in the biomatrix scaffolds (FIGS. 3B, 4D). Amongheterogeneous proteoglycans identified, syndecan was found intercalatedand continuously along the sinusoids, and perlecan is more punctuate inthe space of Disse. The forms of HS-PGs and CS-PGs are found throughoutthe remnants of the sinusoids in the biomatrix scaffolds and in patternscorrelating with the known zonation of the liver tissue.

Proteoglycans and other matrix components are important reservoirs forcytokines and growth factors that bind tightly to their GAGs²⁶. Most ofthe growth factors and hormones are found in the biomatrix scaffoldsnear to the concentrations found in the original tissue. In Table 6 thedata are given from the lysates of rat livers versus rat liver biomatrixscaffolds, and in Table 3, parallel data are provided from human bileduct tissue versus bile duct biomatrix scaffolds. Interestingly, therewere a few examples (e.g., bFGF) that were strongly enriched in liverbiomatrix scaffolds over that found in liver lysates. The growth factorsand cytokines bound are distinct qualitatively and quantitativelybetween the scaffolds of the liver versus bile duct tissue, implicatingeither tissue-specificity or species-specificity. Alternatively, it maybe due, in part, to the fact that the bile duct scaffolds were prepared,from necessity, by shaking the tissue in the buffers on a rocker and notby perfusion through vasculature.

The Chemistry of the Biomatrix Scaffolds Correlates with Histology.

A significant feature of this new protocol is the retention of thematrix chemistry in patterns correlating with the hepatic acinar zones1-3 from portal triad to central vein and with histological entitiessuch as vascular channels and Glisson's Capsule (GC) as shown in FIGS.4A-C. The matrix chemistry peripmially in zone 1 is similar to thatfound in fetal livers and consists, in part, of type III collagen,laminin, and forms of CS-PGs. It transitions to a different matrixchemistry in the mid-acinar (zone 2) and pericentral zones (zone 3)ending with a very stable matrix with high levels of type IV collagenand HP-PGs²⁷.

Myriad proteins (e.g., growth factors and hormones, coagulationproteins, various enzymes) are known to bind to the matrix and to beheld stably via binding to the discrete and specific sulfation patternsin the GAGs or to other matrix components²⁴. Thus, the matrix chemistrytransitions from its start point in the stem cell niche having labilematrix chemistry associated with high turnover and minimal sulfation tostable matrix chemistries and having increasing amounts of sulfationwith progression towards the pericentral zone. We expect that themaintenance of the natural architecture and matrix chemistry correlatingwith histology will facilitate recellularization in tissue engineeringprocesses by guiding cells to specific sites on the biomatrix scaffoldsand/or providing the proper mix of signals to drive expansion and/ordifferentiation into mature cells.

Biomatrix Scaffold can be Prepared from Different Tissues and Species.

The biomatrix scaffolds can be easily prepared from any tissue, normalor diseased and from any species. In FIGS. 13-16 we show biomatrixscaffolds from human pancreas, biliary tree, and duodenum and from ratand porcine pancreas. In FIGS. 5-7 and FIG. 12 are shown effects ofbovine or of rat liver biomatrix scaffolds on hepatic cells. Inaddition, biomatrix scaffolds have been prepared from human abdominalaorta, iliac vein and from rat and pig intestine. Histological,ultrastructural, and immunohistochemical studies on the biomatrixscaffolds indicate a marked tissue specificity, but not speciesspecificity, in their structure, chemical composition, and functions.

Biomatrix Scaffolds Induced and/or Maintained Differentiation of Cells.

Plating hHpSCs onto dishes with sections of liver biomatrix scaffoldsand in HDM tailored for adult liver cells resulted in essentially 100%of the viable cells attached within a few hours onto biomatrixscaffolds; whether intact or after cryogenic pulverization. The coloniesof cells that initially formed on the sections of scaffolds retainedsome of their stem cell phenotype as the cells in the center of thecolonies were able to resist staining with dyes (FIG. 11) and expressedclassic hepatic progenitor markers, such as chemokine (C-X-C motif)receptor 4 (CXCR4) and epithelial cell adhesion molecule (EpCAM) (FIG.5). They divided once or twice and then transitioned into cell cyclearrest and into 3-dimensional (3-D) cord-like morphologies typical forcultures of mature parenchymal cells (FIGS. 5 and 6 for stem celldifferentiation; compare with FIG. 7 and FIG. 12). The HDM used did notrequire all the usual cytokines or growth factors, since these arepresent bound to the biomatrix scaffolds. The transition to growtharrest correlated with staining throughout the colonies with viabilitydyes (FIG. 12), with loss of expression of EpCAM and CXCR4 and with asteady increase in the expression of adult-specific hepatocytic andcholangiocytic genes such as urea and cytochrome P450 3A4. (FIG. 5).

Normal adult rat and adult human hepatocytes were plated onto type Icollagen or on biomatrix scaffolds from rat or bovine livers and intoHDM for adult cells. The adult parenchymal cells were able to attach toscaffolds within 10 minutes (even in serum-free medium) versus withinhours on type I collagen, remained in growth arrest from the point ofattachment; and remained viable and fully functional for more than morethan 8 weeks on scaffolds versus only about 2 weeks on type I collagen.(FIGS. 7 and 12). The levels of functions of the mature liver cells onbiomatrix scaffolds for weeks proved to be the same or similar tofindings of others of freshly isolated, adult hepatocytes²⁸. Thedramatic distinctions are that the cultures on type I collagendeteriorate rapidly after 2 weeks, while those on biomatrix scaffoldsremained stable morphologically and functionally for as long as thecultures were maintained (so far,._,g weeks).

Biomatrix scaffolds contain most of the tissue's extracellular matrixcomponents and matrix-bound cytokines and growth factors providing acomposite set of chemical signals that can be used as an insoluble,stable scaffolding with an extraordinary ability to induce hHpSCs toadult liver fates as well as maintain adult cells fully differentiatedfor weeks. In comparing the extant types of matrix extracts prepared byinvestigators with that of biomatrix scaffold of the present invention,it is clear that physical, enzymatic, and chemical treatments havesubstantial effects on the composition, mechanical behavior, and hostresponses to biological scaffolds derived from the decellularization ofnative tissues and organs, and accordingly, have important implicationsfor their in vitro and in vivo applications. All other existing methodsfor preparation of substrata or scaffolds result in the removal of alarge portion of the matrix components either through the use ofmatrix-degrading enzymes¹⁶ or using buffers that dissolve major portionsof the matrix¹¹. Physical methods (e.g., snap freezing and agitation)can work to prepare matrix extracts from tissues with a layeredstructure such as dermis (e.g., SIS, BSM)²⁹ but are not useful fororgans with complex tissue structures such as liver. By contrast, ourmethod for biomatrix scaffolds resulted in loss of most cellularproteins but preserved essentially all or at least most of the collagensand collagen-associated components including the matrix-bound cytokines,hormones and growth factors.

The extracellular matrix is embedded in a mosaic lipid bilayer, which ineven the simplest organism is a complex, heterogeneous and dynamicenvironment. The delipidation method is a critical facet of theprotocol. The commonly used methods for decellularization of tissuesinvolve ionic detergents such as SDC and sodium dodecyl sulfate (SDS).SDC is relatively milder than SDS, tends to cause less disruption to thenative tissue architecture, and is less effective at solubilizing bothcytoplasmic and nuclear cellular membranes′″. There are no reports oftissue decellularization using SDC alone. Many studies have made use ofa harsh non-ionic detergent (e.g., Triton X-100)3¹ or zwitterionicdetergents (e.g., 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS)³². By contrast, our methodof using a combination of SDC and PLA2 dilapidated the tissue rapidlyand gently.

At least twenty-nine types of collagens (I-XXIX) have been identified sofar in vertebrates with functional roles in cell adhesion,differentiation, growth, tissue development and structuralintegrity^(33,34). The main structural components in the matrix,collagens, are known to remain insoluble in high salt concentrations andat neutral pH^(35,36), a finding that is the basis of our strategy inthe preparation of biomatrix scaffolds. The strategy has the addedadvantages that the collagens enable preservation of the matrixcomponents bound to them, such as laminins and fibronectins (FNs), smallleucine-rich proteoglycans (PGs) and GAGs that in turn preserve thecytokines, growth factors or cell surface receptors that are bound tothem.

The biomatrix scaffolds are unique in their profound ability to inducerapid and consistent differentiation of stem/progenitor cells such ashHpSCs to adult fates and to maintain those lineage-restricted cells andto maintain those lineage-restricted cells or too maintain adult cellsplated onto the scaffolds, as viable and fully functional cells for manyweeks (>8 weeks).

Differentiation of stem cells, such as embryonic stem (ES) cells,induced pluripotent stem (iPS) cells or varying forms of mesenchymalstem cells (MSC) into fully mature liver cell types requires multiplesets of signals (soluble and matrix) presented in stages, with inductionby one set required priming to respond to a different set, and can takemany weeks, up to 6 weeks of culture, to generate cells having the adultliver fate³⁷. Moreover, lineage restriction of MSCs to liver fates givesinconsistent results with adult cells having mixed hepatocyte and MSCphenotypes. The differentiation of ES cells, iPS cells and MSCs resultsin hepatocyte-like cells that express some, but never all, of the majorliver-specific genes; with variability in which genes are observed; andthe protein levels for those hepatic genes expressed are usually low⁴⁰or high for one hepatic gene and negligible for others^(41,42). Bycontrast, the differentiation of the hHpSCs on biomatrix scaffoldsresulted in essentially all of the cells expressing a classic adultphenotype and with urea, albumin and CYP450 activities at levels thatare near normal after a week in culture.

The hepatocyte-like cells from any of these precursors anddifferentiated by protocols other than with biomatrix scaffolds, expresssome, but never all, of the major liver-specific genes, with variabilityin which genes are observed, and with the protein levels for hepaticgenes being usually low or high for one hepatic gene and negligible forothers. For reasons unknown, the results are different from preparationto preparation. It is expected that utilization of the biomatrixscaffolds of this invention should result in more rapid differentiationof these stem cell populations and with greater consistency inachievement of cells with a stable adult phenotype.

Differentiation of determined stem cell populations, such as hHpSCs onbiomatrix scaffolds resulted in essentially all cells expressing aclassic adult repertoire of genes and with urea, albumin, and CYP450activities at near normal levels within 1 to 2 weeks in culture and withstability of that phenotype for many weeks. Thus, the biomatrixscaffolds of the present invention have the potential to greatlyfacilitate differentiation of determined stem cell populations to anadult liver phenotype.

The ability to differentiate stem cells on biomatrix scaffolds toachieve mature and functional cells and tissues offers considerableopportunities for academic, industrial and clinical programs enablingthe use of well differentiated cell types for every type of analyticalstudy, and, most excitingly, enabling the generation of implantable,revascularized tissues or even organs that might be used for basicresearch and clinical programs.

REFERENCES FOR EXAMPLE 1

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Example 2. Methods for Industrial Scale Dispersal of Biomatrix Scaffolds

Additional methods for dispersing the biomatrix scaffolds onto dishesafter pulverization were developed at GigaCyte, LLC (Branford, Conn.).They can be used with biomatrix scaffolds from any tissue includinglarge mammals (e.g., human, porcine and bovine tissues, etc.). Pig andbovine tissues were obtained from a USDA certified meat processingfacility, were transported in RPMI 1640). This medium can be any mediumwith a composition that mimics the constituents of interstitial fluidand with an osmolality that is in the range of 250-350 mOsm/Kg. Thetissues are then processed as described herein to generate biomatrixscaffolds.

These methods include further processing of biomatrix scaffolds forreduction to μm sized particles in suspension for coating plates usingautomation. In one nonlimiting example, the process includeshomogenization of the biomatrix scaffold in high salt solution to ensurecollagens remain insoluble. A modified delipidating buffer comprised of36 units/L phospholipase A2 plus 1% sodium deoxycholate and 0.1 mg/mlsoybean trypsin inhibitor is added at a ratio of 1:1 buffer:homogenate,which results in the buoyant rise of biomatrix material to the top whereit is collected for further processing. The biomatrix material isfurther processed for removal of the delipidating buffer and washed withnuclease solution to remove nucleases: 5 mg/100 ml RNase, 1 mg/100 mlDNase and 0.1 mg/ml soybean trypsin inhibitor. The biomatrix material isthen further processed in high salt (>3.4M NaCl.) buffer to ensureremoval of all residues, to keep the collagens insoluble and also tominimize protease activity. The biomatrix material is further processedto remove the high concentration of salt to prepare for particle sizereduction by washing the biomatrix material in basal medium thatcontains antimicrobic/antimycotics, gentamicin and HEPES buffer. Thebiomatrix material is then further processed to reduce the particle sizeof the biomatrix material to μm sized particles (range 1-100 μm) insuspension, at any dilution, optimally at 1:6, in basal medium thatcontains antimicrobic/antimycotics, gentamicin and HEPES buffer, frozenat −80° C. and then pulverized by cryogenic grinding. If the biomatrixisn't diluted prior to grinding the matrix is too clumpy for coatingplates evenly. This is a key step to allow for grinding and even coatingof plates. The biomatrix particles can be in suspension at any dilutionbut optimally 1:6. The biomatrix particles are thawed and coated ontoplates used for differentiation of stem cells. The biomatrix particlescan be further diluted to any dilution, optimally 1:24 in basal mediumthat contains antimicrobic/antimycotics, gentamicin and HEPES buffer andcoated onto plates used for long term maintenance of primary freshlyisolated, cryopreserved or stem cell-derived terminally differentiatedcell types or for differentiating any stem cell source into hepatocytes.Sterilization of the biomatrix coated plates can be done with, e.g.,gamma irradiation (5,000 rads) and stored at 4° C. (or frozen at −80°C.) until use. Plating cells onto the biomatrix does not require serumcontaining medium for attachment but serum-containing medium can be usedfor applying cells to the biomatrix. The composition of biomatrixparticles from any tissue can be used for coating plates and otherapparati.

As another example, the present invention provides a method of producinga biomatrix scaffold from biological tissue for industrial scaledispersal onto culture apparatus, comprising: a) producing a biomatrixscaffold according to any of the methods of this invention as describedherein, b) diluting the biomatrix scaffold of (a) in basal medium; c)freezing the biomatrix scaffold of (b) at about −80° C.; d) pulverizingthe biomatrix scaffold of (c) by cryogenic grinding into biomatrixparticles ranging in size from about 1 μm to about 100 μm (e.g., about 1μm, 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 95 μm 100 μm, 110 μm, 120 μm, 150 μm, 200 μm,etc.); e) thawing the biomatrix particles of (d) in suspension in basalmedium; and f) dispersing the biomatrix particles of step (e) onto aculture apparatus, thereby producing a biomatrix scaffold frombiological tissue for industrial scale dispersal onto culture apparatus.In some embodiments, this method can further comprise the step ofsterilizing the biomatrix particles, which can be carried out, forexample by gamma irradiation.

In some embodiments of the methods described above, the biomatrixscaffold of step (b) can be diluted at about a 1:6 ratio (e.g., 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.) in basal medium. In someembodiments, the biomatrix particles of step (e) can be diluted at abouta 1:24 (e.g., 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23,1:24, 1:25, 1:26, 1:27: 1:28, 1:29, 1:30, etc) in basal medium.

Outline of Exemplary Protocol Used for Adult Liver 1000× Soybean TrypsinInhibitor

-   -   Final concentration 0.1 mg/ml

Tissue Transport Solution

-   -   Basal medium such as RPMI 1640 or phosphate buffered saline with        calcium and magnesium    -   Antibiotic/Antimycotic 1 OO× solution (Gibco #15240-112)        Gentarnicin (25 ug/ml) (Gibco #15710-072)

Solution 1: Tissue Blending Solution

-   -   3.4M NaCl.    -   0.1 mg/ml Soybean Trypsin Inhibitor (Gibco #17075-029)

Solution 2: Delipidation Solution

-   -   A mix of Solution 1 with 1:1 PBS (w/Ca & Mg); alternative ratios        being 1:2, 1:3, 1:4) or alternatively a basal medium such as        RPMI 1640    -   0.1% Sodium Deoxycholate (Sigma D6750-1OO g)    -   36 u/L Phospholipase A2 (Sigma P9279-25 mg)    -   0.1 mg/ml Soybean Trypsin Inhibitor (Gibco #17075-029)

Solution 3: Nuclease Solution

-   -   Basal medium such as RPMI 1640    -   5 mg/100 ml RNase (Sigma R6513-1 g)    -   1 mg/100 ml DNase (DN25-1 g)    -   0.1 mg/ml Soybean Trypsin Inhibitor

Solution 4: High Salt Solution

-   -   3. M NaCl.

Solution 5: Salt Removal & Grinding Solution

-   -   William's E    -   Antibiotic/Antimycotic (1OO×)    -   Gentamicin (30 ug/ml)

Steps in Biomatrix Isolation Process for Large Organs

-   -   1. Tissue homogenization in high salt    -   2. Decellularization and Delipidation    -   3. Nuclease Removal    -   4. High Salt Wash    -   5. Salt Removal    -   6. Biomatrix Particle Size Reduction    -   7. Coating Plates With Biomatrix

Biomatrix Isolation Process 1.0 Tissue Procurement

-   -   1.1 Obtain organ tissue from a USDA certified and inspected        facility    -   1.2 Transport organ tissue from USDA facility to GigaCyte        laboratory in plastic container in Tissue Transport Solution    -   1.3 Prepare tissue for perfusion or homogenization        -   1.3.1 Homogenization is preferred for in vitro application            in multi-well plates

2.0 Tissue Homogenization

-   -   2.1 Aseptically, cut tissue into small pieces (3×3 cm) with        surgical blade No. 22 and place in Tissue Transport solution to        remove blood    -   2.2 Rinse tissue pieces 3-4 times in Tissue Transport Solution        to remove as much blood as possible    -   2.3 Homogenize 400 g tissue in equal volume of Solution 1 until        mixture is homogeneous (3-5 min.)    -   2.4 Pour homogenized tissue solution into 2 L roller bottle for        decellularization    -   2.5 Repeat for entire batch of tissue pieces

3.0 Decellularization and Delipidation

-   -   3.1 Add equal volume of Solution 2 to homogenized tissue in        roller bottle    -   3.2 Mix well by inverting bottle several times then let set for        15-30 minutes    -   3.3 The buoyant matrix material will rise to the top creating a        distinct separation of light matrix material atop of the liver        colored solution    -   3.4 Pipette off the buoyant matrix material and transfer into        another roller bottle    -   3.5 Pool matrix from multiple homogenizations    -   3.6 Transfer 375 ml matrix material into a 750 ml centrifuge        bottle, add equal volume of Solution 2 to each bottle and shake        vigorously for 1 minute to ensure complete decellularization and        delipidation    -   3.7 Pellet by centrifugation at high (3750 RPM) speed for 10        minutes, then remove supernatant    -   3.8 Repeat this process 1 more times (2 times total)

4.0 Nuclease Removal

-   -   4.1 Remove supernatant after final decellularization wash,        taking care not to disturb the biomatrix pellet    -   4.2 Add equal volume of Solution 3 to each bottle and shake        vigorously for 1 min. to ensure all material is in suspension        and complete removal of nucleases    -   4.3 Pellet by centrifugation at high (3750 RPM) speed for 10        minutes, then remove supernatant    -   4.4 Repeat this process 1 more times. (2 times total)

5.0 High Salt Wash

-   -   5.1 Remove supernatant after final nuclease wash, taking care        not to disturb the biomatrix pellet    -   5.2 Add equal volume of Solution 4 to each bottle and shake        vigorously taking care to ensure all material is in suspension    -   5.3 Pellet by centrifugation at high (3750 RPM) speed for 10        minutes, then remove supernatant        -   5.3.1 At this phase biomatrix doesn't pellet well and is            buoyant after centrifugation.        -   5.3.2 Pipette supernatant off and run through nylon filter            to collect buoyant material        -   5.3.3 Return buoyant matrix collected to the centrifuge            bottle    -   5.4 Repeat this process at least 2 times before stopping        -   5.4.1 Supernatant is slightly tan to clear in color after            final salt wash    -   5.5 After 2° d high salt wash is a good stopping point if it is        late in the day.    -   5.6 Pour the contents of all centrifuge bottles into one roller        bottle and top off with Solution 4 then put biomatrix into 4° C.        over night.    -   5.7 Next day continue remaining salt washes    -   5.8 Shake biomatrix bottle vigorously to mix well biomatrix        material then transfer biomatrix into the centrifuge bottles and        continue 3-4 more salt washes    -   5.9 Condense biomatrix into as few bottles as possible filling        each a maximum of half full to allow for Salt washes

6.0 Salt Removal

-   -   6.1 Remove supernatant after final salt wash taking care not to        disturb the biomatrix pellet    -   6.2 Add equal volume of Solution 5 and shake vigorously to        ensure all biomatrix material is in suspension    -   6.3 Pellet by centrifugation at high (3750 RPM) speed for 10        minutes    -   6.4 Remove supernatant and add equal volume of Solution 5 then        shake vigorously.    -   6.5 Repeat 2 times (3 times total) to ensure supernatant is        clear        -   6.5.1 Biomatrix will pellet more tightly with each spin        -   6.5.2 Supernatant should be clear without any color            following final wash    -   6.6 After final Solution 5 wash record volume of matrix pellets        from each bottle    -   6.7 Transfer and pool all biomatrix pellets into new roller        bottle    -   6.8 Measure volume of biomatrix material    -   6.9 Dilute the pooled biomatrix 1:6 with Solution 5        -   6.9.1 Calculate amount of Solution 5 required            -   6.9.1.1. Example: 200 ml biomatrix×6=1,200            -   6.9.1.2. Add 1 L Solution 5 to 200 ml biomatrix for a                1:6 biomatrix suspension    -   6.10 Aliquot 30 ml biomatrix 1:6 suspension into pre-labeled        freezer bags    -   6.11 Freeze bags of biomatrix at −80° C. and store until        grinding    -   6.12 Prepare “biomatrix lot grinding data sheet” for the new        biomatrix lot        -   6.12.1.1. Record the number of bags produced from this new            batch        -   6.12.1.2. Each bag is a separate grind from the same lot    -   6.13 The biomatrix material produced from this batch is        considered one lot    -   6.14 Assign lot number to the batch

7.0 Biomatrix Particle Size Reduction

-   -   7.1 Prepare the grinding mill (Spex Sample Prep 6870) with LN2        and chill grinding chamber    -   7.2 Remove frozen biomatrix bag from freezer    -   7.3 Break into small pieces    -   7.4 Transfer frozen biomatrix pieces into pre-chilled grinding        mill chamber    -   7.5 Grind 2 rnns (one run is 12×2 minute grinds each followed by        2 minute cool down) 48 minutes each in cryomill    -   7.6 Transfer ground biomatrix into 100 ml bottle pre-labeled        with lot number and bag number (bag number identifies grind        date)    -   7.7 Keep frozen at −80° C. until ready for coating plates

8.0 Preparing Biomatrix for Coating Plates

-   -   8.1 Thaw one bottle of biomatrix rapid thaw method in 37° C.        water-bath    -   8.2 Measure thawed biomatrix suspension (should be 30 ml±2 ml)    -   8.3 Dilute to 1:24 with Solution 5 (see FIG. 17)        -   8.3.1 Calculate volume of Solution 5 to add to the 1:6            biomatrix suspension            -   8.3.1.1. Multiply biomatrix suspension volume×3            -   8.3.1.2. Add that volume to the 1:6 suspension for 1:24                dilution    -   8.4 8.4 Coat plates immediately (letting diluted biomatrix set        will result in clumping)        9.0 Coating Plates with Biomatrix    -   9.1 Determine the number of plates to be coated. Refer to Table        8 for plating volumes        -   9.1.1 Remove plates from packaging aseptically and organize            in the laminar flow hood        -   9.1.2 Transfer appropriate volume of biomatrix suspension            into each well using a multichannel pipettor where            appropriate. Refer to Table 1 for plating volume        -   9.1.3 Ensure that the suspension is evenly distributed            across well-tap plate if necessary but don't remove from            flat surface of hood.        -   9.1.4 Let plates remain undisturbed overnight        -   9.1.5 First thing the next day remove solution from each            well            -   9.1.5.1. Handle plates carefully so matrix coating isn't                disturbed            -   9.1.5.2. Tilt plate toward you without removing plate                edge from hood surface. This stabilizes plate to prevent                jerky movements that will make biomatrix come off the                plate            -   9.1.5.3. Using suction apparatus with fine tip pipette                aspirate entire solution from each well. DO NOT TOUCH                MATRIX SURFACE WITH PIPETTE!!!            -   9.1.5.4. Gently lay plate back down, remove lid and                allow to dry completely (2 hours)            -   9.1.5.5. Moving plate while wet disturbs the biomatrix                coating            -   9.1.5.6. Do not move plates until matrix is dry            -   9.1.5.7. When completely dry replace lid and examine for                quality            -   9.1.5.8. Place plates that have smooth coating into                plate pouch            -   9.1.5.9. Seal pouch and apply label            -   9.1.5.10. Sterilize by gamma irradiation (5,000 rads)            -   9.1.5.11. Store coated plates in 4° C.            -   9.1.5.12. Ship coated plates in packs of 5 on ice pack

10.0 Using Biomatrix Plates

-   -   10.1 Remove plate from packaging aseptically and place in        biological safety cabinet    -   10.2 Add media to each well and place in incubator for at least        2 hours prior to use    -   10.3 When ready to plate cells, remove rehydration media and        wash with tissue culture media once    -   10.4 Add cells

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications,sequences identified by GenBank® Database and/or SNP accession numbers,and other references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

TABLE 1 Molar concentration ranges of NaCl. for Collagen Types 1-V.Molar Concentration range of NaCl. for precipitation (therefore,insolubility at the concentration specified and/or at concentrationsabove that listed) Collagen Type Source Acidic pH Neutral pH I Skin0.7-0.9 2.6 I Trimer Skin 0.7-0.9 4.0 III Skin 0.7-.09 1.5-1.7 IVPlacenta 1.2 1.7-2.0 v Amnion and 1.2 3.6-4.5 chorion; placentaReference: Paul Bornstein and Helene Sage (1980) Structurally DistinctCollagen Types. Annual Review of Biochemistry. 49: 957-1003.

These data are representative of conditions for insolubility of types ofcollagens. One has to identify the collagen types within a tissue andthen use the highest salt concentration identified for those collagensin the tissue and as that for the buffers used for preparing thebiomatrix scaffolds. For example, for skin, one would use a buffer atneutral pH and with a salt concentration of 4.0M, a salt concentrationthat would keep insoluble type I and III collagens. By contrast, forplacenta, one would use a buffer at neutral pH and 4.5M salt to keepinsoluble both type IV and type V collagen.

The types of collagens present in a tissue are distinct at differentages of a host. For example, fetal livers have high levels of type III,IV and V collagens (requiring salt concentrations above 4.0M), whereasadult livers have a mix of type I, III, IV, V, VI and XVIII collagens(requiring lower salt concentrations). Thus, the salt concentrationneeded for a biomatrix scaffold preparation is dictated by therepertoire of collagens that are dominant in the tissue. See reference23 of Example 1 for further details.

TABLE 2 Analyses of Collagen Content in Liver Biomatrix ScaffoldBIOMATRIX SCAFFOLDS {N = 4) LIVER TISSUE (N = 3) Sample Sample SampleSample AVER- Sample Sample Sample AVER- 1 2 3 4 AGE SD 1 2 3 AGE SDAmino Res/ Res/ Res/ Res/ Res/ Res/ Res/ Res/ Res/ Res/ Res/ acids 10001000 1000 1000 1000 1000 1000 1000 1000 1000 1000 Hyp 10.0 13.8 15.312.1 12.8 2.3 0.0* 0.0* 0.0* 0.0* 0.0* Asp .82.7 77.1 78.4 85.5 80.9 3.993.0 94.5 90.8 92.8 1.8 Thr 52.6 51.1 45.6 52.4 50.4 3.3 52.2 51.0 53.752.3 1.3 Ser 56.5 53.7 61.9 62.4 58.6 4.2 57.7 57.0 62.0 58.9 2.7 Glu112.0 107.0 117.4 118.1 113.6 5.2 123.9 122.0 130.1 125.4 4.2 Pro 52.352.2 55.7 51.6 52.9 1.9 46.6 45.9 46.4 46.3 0.4 Gly 118.7 134.0 125.4109.4 121.9 10.4 88.2 84.6 89.1 87.3 2.4 Ala 88.3 86.1 89.5 83.6 86.92.6 79.0 78.2 90.6 82.6 7.0 Val 64.3 57.2 54.8 65.9 60.6 5.4 71.6 71.370.1 71.0 0.8 Met 21.7 20.6 20.7 20.4 20.8 0.6 21.4 21.4 21.7 21.5 0.2lie 51.8 47.7 47.4 41.7 47.2 4.2 49.6 50.1 42.3 47.3 4.3 Leu 92.7 83.8109.7 87.5 93.4 11.5 93.8 95.1 92.7 93.9 1.2 Tyr 30.8 26.9 22.4 28.127.0 3.5 31.8 32.1 27.5 30.5 2.6 Phe 45.5 40.7 42.4 42.8 42.8 2.0 47.851.0 43.7 47.5 3.7 His 21.0 18.8 13.2 21.8 18.7 3.9 21.8 21.5 24.1 22.51.4 Hyl 1.0 1.8 1.6 4.3 2.2 1.5 0.0** 0.0** 0.0** 0.0** 0.0** Lys 40.670.0 49.8 66.2 56.7 13.8 74.6 78.5 73.0 75.3 2.8 Arg 57.5 57.6 48.7 46.152.5 6.0 47.0 46.0 42.1 45.0 2.6 Note: *less than 0.2 res/1,000; **notdetected.

TABLE 3 Analyses of Growth Factors Bound to Bile Duct BiomatrixScaffolds Human Bile Duct Human Bile Biomatrix NAME CYTOKINE FULL NAMEDucts Scaffolds % bFGF Basic fibroblast growth factor 58299 126 0% b-NGFNerve growth factor (beta polypeptide) 516 81 16% EGF Epidermal growthfactor 91 108 119% EGF R Epidermal growth factor receptor 479 145 30%FGF-4 Fibroblast growth factor-4 31 36 116% FGF-6 Fibroblast growthfactor-6 14 17 121% FGF-7 Fibroblast growth factor-? 149 23 15% GCSFGranulocyte-colony stimulating Factor 207 233 113% GDNF Glial-derivedneurotrophic factor 53 49 92% GM-CSF Granulocyte macrophage-colony 10897 90% stimulating factor HB-EGF Heparin-binding epidermal growth 28 2382% factor IGFBP-1 Insulin-like growth factor binding 431 61 14%proteins 1 IGFBP-2 Insulin-like growth factor binding 255 20 8% proteins2 IGFBP-3 Insulin-like growth factor binding 77 54 70% proteins 3IGFBP-4 Insulin-like growth factor binding 81 58 72% proteins 4 IGFBP-6Insulin-like growth factor binding 783 107 14% proteins 6 JGF-1Insulin-like growth factor-I 18 6 33% IGF-1 SR Insulin-like growthfactor-I 89 25 28% IGF-11 Insulin-like growth factor-2 2873 3945 137%M-CSF Macrophage-colony stimulating factor 149 105 70% M-CSF RMacrophage colony stimulating factor 358 71 20% receptor NT-3Neurotrophin-3 71 71 100% NT-4 Neurotrophin-4 75 58 77% PDGF R aPlatelet-derived growth factor receptor 104 63 61% alpha PDGF R bPlatelet-derived growth factor receptor 489 110 22% beta PDGF-AAPlatelet-derived growth factor AA 114 52 46% PDGF-AB Platelet-derivedgrowth factor AB 87 65 75% PDGF-BB Platelet-derived growth factor BB 15575 48% PIGF Phosphatidylinositol glycan anchor 146 14 10% biosynthesis,class F SCF Stromal cell-derived factor-1 39 22 56% SCF R Strama!cell-derived factor receptor 159 31 19% TGF-a Transforming growth factoralpha 52 25 48% TGF-b Transforming growth factor-beta 234 277 118% TGF-b2 transforming growth factor-beta 2 103 121 117% TGF-b 3 Transforminggrowth factor-beta 3 28 16 57% VEGF Vascular endothelial growth factor74 35 47% VEGF R2 Vascular endothelial growth factor 108 33 31% receptor2 VEGF R3 Vascular endothelial growth factor 45 40 89% receptor 3

TABLE 4 Antibodies Utilized Host and Antibody's name isotype CompanyCatalog NO. Dilution Primary Antibodies to extracellular matrixcomponents 1. collagen 1 mouse lgG1 Sigma C2456 1/2000 2. collagen 3A1Mouse lgG1 Sigma C7805 1/2000 3. collagen 4A1 Goat lgG Santa Cruzsc-9302 1/50 4. collagen 5A1 Rabbit lgG Santa Cruz sc-20648 1/50 5.collagen 6 Rabbit lgG Santa Cruz Sc-20649 1/50 6. chondroitin mouse lgMSigma C8035 1/200 sulfate 7. Elastin Rabbit lgG Abeam ab21610 1/200 8.entactin Rat lgG GeneTex GTX72367 1/200 (nidogen 1) 9. heparan Mouse lgMSeiko, Japan 270426 1/200 sulfate 10. HS-PG: Mouse NeoMarkers RT-7941/200 perlecan lgG2a 11. HS-PG: Goat lgG R&D AF2780 1/100 syndecan-1 12.Fibronectin Mouse lgG1 Sigma F7387 1/200 13. Laminin Mouse lgG1 SigmaL8271 1/1000 Primary Antibodies to other proteins 1. AFP Rabbit lgGNovus NB100-1611 1/200 Biologicals 2. Albumin Rabbit lgG Novus NB600-570 1/200 Biologicals 3. ASMA mouse lgG2a Sigma A2547 1/300 4. Ck18mouse lgG2b Sigma SAB3300016 1/400 5. hCK19 mouse lgG2a Abeam ab77541/250 6. CK19 Rabbit lgG Abeam Ab52625 1/200 7. CYP3A4 Mouse lgG AbnovaH00001576- 1/200 B01P 8. Desmin Rabbit lgG Abeam Ab8592 1/200 9. EpCAMMouse lgG1 NeoMarkers MS-181 1/200 10. secretin Rabbit lgG Santa Cruzsc.:.26633 1/100 receptor 11. PAN CK Mouse lgG1 Abeam ab-7753 1/300 12.Tubulin-a Mouse lgG1 Neomarkers MS-581 1/1000 13. Vimentin Mouse lgG1Abeam Ab8978 1/200 Secondary Antibodies or dyes for florescent cellstain or tissue immunohistochemistry 1. Alexa Fluor ® 488/594 goat anti-Molecular 1/500 mouse lgG 1 or 2a or anti rabbit lgG Probes 2.VECTASTAIN ® ABC system Vector Laboratories 3. NovaRED ™ SUBSTRATE KITVector Laboratories 4. Phalloidin 488 Molecular 1/500 Probes

TABLE 5 Primers Utilized for RT-PCR GenBank Amplicon No Name Full nameAccession Sequence (5′→3′) Tm Size  1 CXCR-4 chemokine (C-X- AJ224869TACACCGAGGAAATGGGCTC 63 112 C motif) A receptor 4 AGATGATGGAGTAGATGGTG60.4 GG  2 EpCAM epithelial cell NM ATAACCTGCTCTGAGCGAGTG 61.6 104adhesion 002354 TGAAGTGCAGTCCGCAAACT 62.3 molecule  3 KRT19 keratin 19NM ACCAAGTTTGAGACGGAACA 60.2 181 002276 G CCCTCAGCGTACTGATTTCCT 61.3  4HNF6 hepatocyte NM ATGTGGAAGTGGCTGCAGGA 60.7 105 nuclear factor 6,004498 TGTGTTGCCTCTATCCTTCCC 61.2 alpha  5 FOXA2 forkhead box A2 NMGCGACCCCAAGACCTACAG 61.7 162 021784 GGTTCTGCCGGTAGAAGGG 61.7  6 PROX1prospero NM TTGACATTGGAGTGAAAAGG 61 100 homeobox 1 002763 ACGTGCTCAGAACCTTGGGGATTC 61.8  7 AFP alpha-fetoprotein NMCTTGCACACAAAAAGCCCAC 61.9 138 001134 T GGGATGCCTTCTTGCTATCTC 61.8 AT  8ALB albumin M12523 TTTATGCCCCGGAACTCCTTT 61.4 90 ACAGGCAGGCAGCTTTATCA62.4 G  9 TF transferrin NM CCTCCTACCTTGATTGCATCA 60.2 137 001063 GTTTTGACCCATAGAACTCTGC 60 C 10 CYP3A4 cytochrome NM AAGTCGCCTCGAAGATACAC60.9 174 P450, family 3, 017460 A subfamily A, AAGGAGAGAACAC 61.7polypeptide 4 TGCTCGTG 11 TAT tyrosine NM TTTGGGACCCTGTACCATTGT 61 102aminotransferase 000353 GCATTGGACTTGAGGAAGCT 61 C 12 G6PC glucose-6- NMTCAGGGAAAGATAAAGCCGA 61.8 105 phosphatase, 000151 CC catalytic subunitAGGTAGATTCGTGACAGACA 61.1 GAC 13 CFTR cystic fibrosis NMAAAAGGCCAGCGTTGT CTCC 63 170 transmembrane 000492 TGAAGCCAGCTCTCTATCCCA62.1 conductance regulator 14 GGT1 gamma- J05235 GGGGAGATCGAGGGCTATGA 63150 glutamyl- G transferase 1 GATGACGGTCCGCTTGTTTTC 61.8 15 AE2 SLC4A2NM GCCAAGGGCGCAGATTCTT 63 103 003040 CCAGGGTGCGGTGAAGTTC 62.9 16 ASSTSLC10A2 NM TGTGTTGGCTTCCTCTGTCAG 62 115 000452

TABLE 6 Analyses of Growth Factor Bound to Liver Biomatrix Scaffolds RatRat Biometrix Name Cytokine Full Name Livers Scaffolds Percent bFGFBasic fibroblast growth 100.06 394.14 394 factor EGF Epidermal grov.rth74.81 76.02 102 factor EGF R Epidermal gro'Nth 92.81 81.64 88 factorreceptor ffiF-4 Fibroblast growth 15.06 1321 88 factor-4 ffiF·6Flbroblast gro11.rth 4.81 3.77 78 factor-6 FGF-7 Fi broblast gro'Nth10.06 6.32 63 factor-7 GCSF Granulocyte-colony 348.06 338.20 97stimulating factor GDNF Glial-derivecl 81.31 43.59 54 neurotrophicfactor GM-CSF Granulocyte 133.56 105.38 79 macrophage-colorrfstimulating factor HB-EGF Heparin-binding 44.56 38.23 86 epidermalgrowth factor lGFBP-1 lnsulin·Hke growth 67.81 70.40 104 factor bindingproteins 1 fGFBP-3 lnsultn-like grOwth 140.81 201.90 143 factor bindingproteins 3 IGFBP-4 lnsulin-like growth 83.56 58.92 71 factor bindingproteins 4 IGFBP-6 Insulin-I ike growth 91.81 72.19 79 factor bindingproteins 6 !GF-! Insulin-like growth 1.56 1.98 127 factor-I !GF-1 SRInsulin-like growth 7.31 3.51 48 factor-I IGF·U Insulin-like growth3749.06 3482.52 93 factor-2 M-CSF Macrophage-colony 170.31 134.68 79stimulating factor M-CSF R Macrophage colony 70.56 50.47 72 stimulatingfactor receptor NT.J Neurotmphin--3 25.56 5.03 20 NT-4 Neurotrophin-455.06 43.59 79 PDGF R a Platelet-derived 10.56 21.11 200 growth factorreceptor alpha PDGF R b Platelet-derived 113.81 85.46 75 growth factorreceptor beta PDGF-AA Platelet-derived 62.06 106.40 171 growth factor AAPDGF-AB Platelet-derived 19.31 19.34 100 gro11.rth factor AB PDGF-BBPlatelet·derived1owth 9.56 1423 149 factor BB PIGF Phosphatidyl1nositol4.81 8.36 174 glycan anchor biosynthesis, class F SCF Stromalcell-derived 2.06 42.56 2064 factor-1 SCF R Stromal cell-derived 17.0617.80 104 factor receptor TGF-a Transforming growth 21.31 21.63 102factor alpha TGF-b Transforming growth 330.31 342.77 104 fuctor-betaTGF-b 2 transforming growth 134.06 152.34 114 factor-beta 2 TGF-b 3Transforming growth 1.06 0.18 17 factor-beta 3 VEGF Vascular endothelial70.56 94.14 133 growth factor VEGF R2 Vascular endothelial 13.56 11.9388 growth factor receptor 2 VEGF R3 Vascular endothelial 459.56 46.91 10growth factor receptor 3

TABLE 7 Properties of human hepatic stem cells (hHpSCs) after isolationand in culture Self-Replication Conditions illay DifferentiationConditions (Day 12) 12) DM and hHpSCs- KM and TCP or DM and Type IBiomatrix Properties freshly Isolated Type ID collagen CollagenScaffolds How long to attach — 4-5 hours on 7-12 hours 3 hours TCP; -3hours on type III collagen % attachment of — 60-80% on TCP 100% viablecells and -100% on type III collagen Morphology of 2-dimensional Cordsof cells; Cords of cells; colonies (monolayer) somewhat very 3- coloniescuboidal dimensional Doubling time — A division every A division everyOnly a few (division rate) 36 hrs on TCP 40-SO hours divisions and 24-26hours transitioning to transitioning to on type III growth arrest bygrowth arrest by s collagen 7-10 days days Duration of >6 months -2weeks >8 weeks as Viability (remaining as differentiated stem cells)cells (not tested yet for longer) Percentage of Cells Expressing theSpecified Marker EpCAM 100% Present on membranes of smallcholangiocytes; no expression at all in mature hepatocytes NCAM >80%None CD133/1 90% None SOX 17 100% None CK 8/18, E- 100% 100% cadherinCK19 90% Present in cholangiocytes but not in hepatocytes a-fetoproteinNone; Ifany, then due to Moderate Expression in contamination withhepatoblasts expression in first 2-3 days, most cells at 10-12 decreasesdays dramatically thereafter, no expression from day 7 on. P450s None ornegligible levels 100% (−18,000 100% (35,000 RLU)* RLU)* Ureasynthesized None 2.S mgs/dL 7 mgs/dL Markers: mature Most of the cellshave none; those albumin, transferrin protein, tyrosine hepatocytesexpressing do so weakly (e.g. aminotransferase (TAT), glycogen.albumin); all express transferrin Weak levels on collagen I versus mRNAbut no transferrin protein strong on biomatrix scaffolds Markers: matureMost none; those expressing any Secretin receptor, AE2, ABAT,cholangiocytes do so weakly: (e.g. CFTR, GGTI, CFTR, GGTl, AE2, ASBT,but with AE2: no expression of ASBT and weak levels on collagen I versusvery late aquaporins strong on biomatrix scaffolds KM = Kubota's Medium,a serum-free medium used for hHpSCs and progenitors; HDM-L =differentiation medium derived from KM and with hormones and factorsgiven in the methods. TCP = tissue culture plastic. *See Figure SF;**See Figure SE

TABLE 8 Biomatrix plating volumes for multiwall plates Plate SizeVolume/well Volume/Plate Plates/Bottle 384-well  30 ul 12.0 ml  1096-well  50 ul 5.0 ml 24 24-well 300 ul 7.5 ml 16 12-well 800 ul  10 ml12 6-well  1.5 ml   9 ml 13

1-19. (canceled)
 20. A method of producing a plurality of biomatrixparticles from a biological tissue for dispersal onto culture apparatus,comprising: (a) perfusing or homogenizing the biological tissue with abuffer comprising a salt concentration from about 3.5 M NaCl to about4.5 M NaCl; (b) perfusing the biological tissue or extracting thehomogenate of step (a) with a delipidating buffer comprising lipases anddetergents in a serum free first medium, having a neutral pH and anosmolality from about 250 mOsm/kg to about 350 mOsm/kg, wherein thedetergents do not comprise sodium dodecyl sulfate or TritonX-100; (c)perfusing the biological tissue or extracting the homogenate of step (b)with a buffer at a neutral pH and comprising a salt concentrationranging from about 2.0 M NaCl to about 5.0 M NaCl, the concentrationchosen to not lose insoluble collagens identified in the biologicaltissue; (d) perfusing the biological tissue or extracting the homogenateof step (c) with RNase and DNase in a buffer; (e) rinsing the biologicaltissue or homogenate of step (d) with a second medium having a neutralpH, is serum-free and having an osomolality ranging from about 250mOsm/kg to about 350 mOsm/kg, thereby producing an intact or homogenizedbiomatrix scaffold from the biological tissue; (f) pulverizing theintact or homogenized biomatrix scaffold into a plurality of biomatrixparticles ranging in size from about 1 nm to about 100 μm.
 21. Themethod of claim 20, wherein the intact or homogenized biomatrix scaffoldcomprises at least 95% of the collagens and most collagen-associatedmatrix components, matrix bound growth factors, hormones, and cytokinesof the biological tissue.
 22. The method of claim 20, wherein thebiological tissue is selected from the group consisting of liver, lung,thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliarytree, duodenum, abdominal aorta, iliac vein, heart and intestines. 23.The method of claim 20, wherein the pulverizing of step (f) comprisescryogenic grinding.
 24. The method of claim 20, wherein the detergentsare selected from the group consisting of salts of deoxycholic acid,1-heptanesulfonic acid, N-laurylsarcosine, lauryl sulfate, 1-octanesulfonic acid and taurocholic acid; benzalkonium chloride;cetylpyridinium; methylbenzethonium chloride; decamethonium bromide;alkyl betaines; alkyl amidoalkyl betaines;N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;phosphatidylcholine; n-decyl α-D-glucopyranoside; n-decylβ-D-maltopyranoside; n-dodecyl β-D-maltoside; n-octylβ-D-glucopyranoside; sorbitan esters; n-tetradecyl β-D-maltoside;tritons; Nonidet-P-40; Poloxamer 188; sodium lauryl sulfate; and sodiumdeoxycholate.
 25. The method of claim 20, wherein the lipases arephospholipases.
 26. The method of claim 20, wherein the serum free firstmedium is a basal medium selected from the group consisting of RPMI1640, DME/F12, DME, F12, Waymouth's, and William's medium.
 27. Themethod of claim 20, wherein the second medium comprises at least one ofthe constituents present in interstitial fluid.
 28. The method of claim20, wherein the delipidating buffer of step (b) comprises from about 20units/L to about 50 units/L phospholipase A2 and about 1% sodiumdeoxycholate in the first medium.
 29. The method of claim 20, whereinthe salt concentration of the buffer of step (c) is from about 3.4 MNaCl to about 3.5 M NaCl when used for scaffold preparation from anadult liver and is from about 4.0 M NaCl to about 4.5 M NaCl when usedfor scaffold preparation from a fetal liver.
 30. The method of claim 20,wherein the buffer of step (c) further comprises a protease inhibitor.31. The method of claim 31, wherein the protease inhibitor is soybeantrypsin inhibitor.
 32. The method of claim 20, wherein the buffer ofstep (d) further comprises a protease inhibitor.
 33. The method of claim32, wherein the protease inhibitor is soybean trypsin inhibitor.
 34. Themethod of claim 20, wherein all media and buffers of steps (a) through(e) are free of a detectable amount of an enzyme that degradesextracellular matrix components.
 35. The method of claim 20, wherein thebiological tissue is from a mammal.